Method for non-invasive monitoring of fluorescent tracer agent with diffuse reflection corrections

ABSTRACT

A method of monitoring a time-varying fluorescence signal emitted from a fluorescent agent from within a medium with time-varying optical properties is provided that includes providing a measurement data set that includes a plurality of measurement entries that include at least two measurements obtained from a patient before and after administration of the fluorescent agent. The measurements may include one or more of: a DRex signal detected by an unfiltered light detector during illumination by excitatory-wavelength light from first region adjacent to the diffuse reflecting medium; a Flr signal detected by a filtered light detector during illumination by excitatory-wavelength light; and a DRem signal detected by the unfiltered light detector during illumination by emission-wavelength light. The method further includes identifying a post-agent administration portion of the measurement data set; and transforming each Flr signal to an IF signal representing a detected fluorescence intensity emitted solely by the fluorescent agent.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/452,025 filed Jan. 30, 2017, which is incorporated herein in itsentirety.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to methods for non-invasivemonitoring of a fluorescent tracer agent within a medium characterizedby scattering and/or absorption of light. More particularly, the presentdisclosure relates to methods for non-invasive assessment of kidneyfunction by monitoring the clearance of an exogenous fluorescent tracerwithin the tissues of a patient in vivo.

Dynamic monitoring of renal function in patients at the bedside in realtime is highly desirable in order to minimize the risk of acute renalfailure brought on by various clinical, physiological and pathologicalconditions. It is particularly important in the case of critically illor injured patients because a large percentage of these patients facethe risk of multiple organ failure (MOF) incited by one or more severedysfunctions, such as: acute lung injury (ALI), adult respiratorydistress syndrome (ARDS), hypermetabolism, hypotension, persistentinflammation, and/or sepsis. Renal function may also be impaired due tokidney damage associated with administration of nephrotoxic drugs aspart of a procedure such as angiography, diabetes, auto-immune disease,and other dysfunctions and/or insults causally linked to kidney damage.In order to assess a patient's status and to monitor the severity and/orprogression of renal function over extended periods, there existsconsiderable interest in developing a simple, accurate, and continuousmethod for the determination of renal failure, preferably bynon-invasive procedures.

Serum creatinine concentration, an endogenous marker of renal function,is typically measured from a blood sample and used, in combination withpatient demographic factors such as weight, age, and/or ethnicity toestimate glomerular filtration rate (GFR), one measure of renalfunction. However, creatinine-based assessments of renal function may beprone to inaccuracies due to many potential factors, including: age,state of hydration, renal perfusion, muscle mass, dietary intake, andmany other anthropometric and clinical variables. To compensate forthese variances, a series of creatinine-based equations (most recentlyextended to cystatin C) have been developed which incorporate factorssuch as sex, race and other relevant factors for the estimation ofglomerular filtration rate (eGFR) based on serum creatininemeasurements. However, these eGFR equations are not provided with anymeans of compensating for most of the above sources of variance, andtherefore have relatively poor accuracy. Further, the eGFR methodtypically yields results that lag behind true GFR by up to 72 hrs.

Exogenous marker compounds, such as inulin, iothalamate, ⁵¹Cr-EDTA,Gd-DTPA and ^(99m)Tc-DTPA have been used in existing methods formeasuring GFR. Other endogenous markers, such as ¹²³I and ¹²⁵I labeledo-iodohippurate or ^(99m)Tc-MAG3 have been used to in other existingmethods for assessing the tubular secretion process. However, the use oftypical exogenous marker compounds may be accompanied by variousundesirable effects including the introduction of radioactive materialsand/or ionizing radiation into the patient, and laborious ex vivohandling of blood and urine samples, rendering existing methods usingthese exogenous markers unsuitable for real-time monitoring of renalfunction at a patient's bedside.

The availability of a real-time, accurate, repeatable measure of renalexcretion rate using exogenous markers under patient-specific yetpotentially changing circumstances would represent a substantialimprovement over any currently practiced method. Moreover, a method thatdepends solely on the renal elimination of an exogenous chemical entitywould provide a direct and continuous pharmacokinetic measurementrequiring less subjective interpretation based upon age, muscle mass,blood pressure, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIG. 1 is a schematic illustration of a single-wavelength renalmonitoring device in one aspect;

FIG. 2 is a schematic illustration of a dual-wavelength renal monitoringsystem in one aspect;

FIG. 3 is a graph summarizing the absorption, transmission, and emissionspectra of various devices, materials, and compounds associated with thenon-invasive monitoring of an exogenous fluorescent agent in vivodefined over light wavelengths ranging from about 430 nm to about 650nm;

FIG. 4 is a graph summarizing the absorption spectra of oxyhemoglobin(HbO₂) and deoxyhemoglobin (Hb) defined over light wavelengths rangingfrom about 200 nm to about 650 nm;

FIG. 5 is a schematic illustration of the timing of light pulse cyclesassociated with data acquisition by a dual-wavelength renal monitoringsystem in one aspect, in which each light pulse cycle includes lightpulses produced at the excitation wavelength and at the emissionwavelength in sequence;

FIG. 6 is a side view of a sensor head of a renal function monitoringsystem in one aspect;

FIG. 7 is a bottom view of the sensor head of FIG. 6;

FIG. 8 is a top interior view of the sensor head of FIG. 6 illustratingan arrangement of various electrical components within a housing of asensor head of a renal function monitoring system in one aspect;

FIG. 9 is an enlargement of the interior view of FIG. 8;

FIG. 10 is a schematic illustration of the apertures formed within acontact surface of a sensor head of a renal function monitoring systemin one aspect;

FIG. 11 is a schematic illustration of synchronous detection of light bya light detector of a sensor head in one aspect;

FIG. 12 is a schematic illustration of light signal modulation anddemodulation by the sensor head in one aspect;

FIG. 13 is a block diagram illustrating the subunits of a processingunit in one aspect;

FIG. 14A is a flow chart illustrating the steps of a global errormapping method of determining the parameters of a diffuse reflectancecorrection equation in one aspect;

FIG. 14B is a flow chart illustrating the steps of a global errormapping method of determining the parameters of a diffuse reflectancecorrection equation in a second aspect;

FIG. 15A is a graph of representative intrinsic fluorescencemeasurements of the fluorescent agent (IF_(agent)) detected by a renalmonitoring device obtained before and after injection of an exogenousfluorescent agent. A subset of the data selected for analysis todetermine correction factors are shown highlighted in orange.

FIG. 15B is a graph of representative intrinsic fluorescencemeasurements of the fluorescent agent (IF_(agent)) detected by a renalmonitoring device obtained before and after injection of an exogenousfluorescent agent. A subset of the data selected for analysis by fittingthe IF_(agent) to a plasma-derived IF_(agent) to determine correctionfactors are shown highlighted in orange.

FIG. 16 is a graph comparing the log-transformed single-exponentialcurve fit of the corrected fluorescence signal measurements (log [Fit],black dashed line) and the corrected fluorescence signal measurementsfrom FIG. 15 (IF_(agent), red line) over a portion of the selectedanalysis region from FIG. 15.

FIG. 17 is a map of a representative error surface summarizingnormalized root-mean-square errors (RMSE, color scale) calculated forthe difference between the linear fit to the log of the fluorescence andthe corrected fluorescence signal measurements for a range of correctionfactors k_(ex) and k_(em,filtered), with a minimum RSME regionidentified by a white arrow overlaid on the map;

FIG. 18 is a graph comparing raw (F, blue line) fluorescence signalmeasurements and corrected (IF, red line) fluorescence signalmeasurements obtained before and after injection of an exogenousfluorescent agent.

FIG. 19 is a flow chart summarizing the steps of a linear regressionmodel method of determining the parameters of a diffuse reflectancecorrection equation in one aspect;

FIG. 20 is a graph of log-transformed raw fluorescence signal (Log(Flr))showing the regions of the data used as project fits for: a linearregression model used to develop a data correction algorithm (orangeline), the response variable for the linear regression model (blackdashed line), and the region of highly varying data used to train thelinear regression model (blue line);

FIG. 21A is a graph of raw fluorescence signal measurements obtainedbefore and after injection of an exogenous fluorescent agent.Measurements of raw fluorescence signals were obtained during exposureto various perturbations denoted as colored regions starting at about13:50 hours. The various perturbations included variations in bloodoxygenation in the test subject, application and removal of pressure tothe measured region, administration of blood pressure medication to thetest subject, cooling of the measured region, and removal/replacement ofthe sensor head of the device;

FIG. 21B is a graph of corrected fluorescence signal measurements ofFIG. 21A;

FIG. 21C is a graph of diffuse reflectance signal measurements measuredsimultaneously with the raw fluorescence signal measurements of FIG.21A. These signals are used in the correction of the raw fluorescencesignal measurements of FIG. 21A to produce the corrected signal shown inFIG. 21B;

FIG. 22A is a block diagram illustrating a plurality of modules of apre-processing subunit in one aspect;

FIG. 22B is a block diagram illustrating a plurality of modules of apre-processing subunit in a second aspect;

FIG. 23 is an isometric view of a sensor head of a renal functionmonitoring system in a second aspect;

FIG. 24 is a bottom view of the sensor head of a renal functionmonitoring system illustrated in FIG. 23;

FIG. 25 is an isometric view of the sensor head of a renal functionmonitoring system illustrated in FIG. 23 with the upper housing andvarious electrical components removed to expose an inner housing; and

FIG. 26 is an exploded view of the inner housing of the sensor headillustrated in FIG. 25.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein may be usedin the practice or testing of the present disclosure, the preferredmaterials and methods are described below.

A sample, as used herein, refers to a single, discrete data valueacquired from a signal and/or telemetry analog-to-digital converter(ADC) for a single acquisition/telemetry channel.

A measured value, as used herein, refers to a single, discrete datavalue created by demodulating or accumulating a sequence of samples fromone acquisition channel.

A measurement, as used herein, refers to a set comprising theDemodulated In-Phase, Demodulated Out-of-Phase, and Averaged measurementvalues from one acquisition channel.

A measurement subset, as used herein, refers to a set comprising allmeasurements for all acquisition channels during a single source LEDillumination. For example, all measurements of an acquisition channelmay include demodulated in-phase, demodulated out-of-phase, and averagedmeasurements.

A measurement set, as used herein, refers to a set comprising onemeasurement subset for each source LED.

An acquisition, as used herein, refers to the overall process by which ameasurement set is obtained.

A measurement sequence, as used herein, refers to a sequence of one ormore measurement sets.

A telemetry value, as used herein, refers to a single, discrete datavalue acquired from a single channel of a telemetry ADC.

A telemetry set, as used herein, refers to a set comprising onetelemetry value from each telemetry channel.

FIG. 1 is a schematic illustration of a system 100, provided as anon-limiting example, in which fluorescence 102 with an emissionwavelength (λ_(em)) is detected from a region of interest of a patient104 using a light detector 110 configured to detect only those photonswith an emission wavelength (λ_(em)). In general, the exogenousfluorescent agent 112 produces fluorescence 102 in response to anexcitation event including, but not limited to: illumination by light106 at an excitation wavelength (λ_(ex)), occurrence of an enzymaticreaction, changes in local electrical potential, and any other knownexcitation event associated with exogenous fluorescent agents. In anaspect, the system 100 may include a light source 108 configured todeliver light 106 at an excitation wavelength (λ_(ex)) to the patient104. In this aspect, the fluorescence 102 is produced in response toillumination by the light 106. In addition, the excitation wavelength(λ_(ex)) of the light 106 and the emission wavelength (λ_(em)) of thefluorescence 102 are spectrally distinct (i.e., λ_(ex) is sufficientlydifferent from λ_(em)) so that the light detector 110 may be configuredto selectively detect only the fluorescence 102 by the inclusion of anyknown optical wavelength separation device including, but not limitedto, an optical filter.

In some aspects, changes in the fluorescence 102 may be monitored toobtain information regarding a physiological function or status of thepatient. By way of non-limiting example, the time-dependent decrease inthe fluorescence 102 measured after introduction of the exogenousfluorescent agent 112 into a circulatory vessel of the patient 104 maybe analyzed to obtain information regarding renal function of thepatient 104. In this non-limiting example, the rate of decrease influorescence 102 may be assumed to be proportional to the rate ofremoval of the exogenous fluorescent agent 112 by the kidneys of thepatient 104, thereby providing a measurement of renal functionincluding, but not limited to: renal decay time constant (RDTC) andglomerular filtration rate (GFR).

Without being limited to any particular theory, the intensity offluorescence 102 detected by the light detector 110 may be influenced byany one or more of numerous factors including, but not limited to: theintensity or power of the light 106 at λ_(ex) delivered to the patient104, the scattering and absorption of the light 106 passing throughintervening tissues 114 of the patient 104 between the light source 108and the exogenous fluorescent agents 112, the concentration of exogenousfluorescent agents 112 illuminated by the light 106, and the scatteringand absorption of the fluorescence 102 at λ_(em) passing throughintervening tissues 114 of the patient 104 between the exogenousfluorescent agents 112 and the light detector 110.

Existing methods typically assume that the optical properties within theintervening tissue 114 remain essentially unchanged throughout theperiod during which measurements are obtained by the system 100. As aresult, existing methods typically obtain initial measurements throughthe intervening tissue 114 of the patient 104 prior to introduction ofthe exogenous fluorescent agent 112, and these initial measurements aresubtracted to correct all subsequent data obtained after introduction ofthe exogenous fluorescent agent 112. However, during long-termmonitoring of the patient 104, changes in the optical properties of theintervening tissue 114 may occur due to changes in at least onecharacteristic including, but not limited to: optical couplingefficiency of the light detector 110 to the patient 104; concentrationof chromophores such as hemoglobin due to changes in blood volume causedby vascular dilation, constriction, or compression; changes in theoptical properties of chromophores such as hemoglobin due to changes inoxygenation status; and changes in tissue structure such as changesrelated to edema.

These dynamic changes in the optical properties of the interveningtissue 114 may introduce uncertainty into long-term measurements offluorescence 102. By way of non-limiting example, changes in the opticalproperties of the intervening tissue 114 may modulate the intensity orpower of the light 106 illuminating the exogenous fluorescent agents112, causing a modulation of the fluorescence 102 produced by theexogenous fluorescent agents 112 that may be erroneously interpreted asa modulation in the concentration of the exogenous fluorescent agents112. By way of another non-limiting example, changes in the opticalproperties of the intervening tissue 114 may modulate the intensity orpower of the fluorescence 102 reaching the light detector 110 that mayalso be erroneously interpreted as a modulation in the concentration ofthe exogenous fluorescent agents 112. The potential modulation ofchanges in the optical properties of the intervening tissue 114 mayintroduce uncertainty into measurements of fluorescence 102, inparticular those measurements associated with long-term monitoring offluorescence 102 as described herein above.

In various aspects, a method of correcting in vivo real-timemeasurements of fluorescence from an exogenous fluorescent agent toremove the effects of changes in the optical properties within thetissue of the patient is provided. The inclusion of an additionalmeasurement of light passing through the tissue of the patient via aseparate optical pathway (i.e. diffuse reflectance) from the opticalpathway of the fluorescence measurements enhanced the quantification ofchanges in the optical properties of the tissue during prolongedmonitoring of fluorescence from an exogenous fluorescent agent within apatient. The inclusion of this additional measurement in the correctionmethod in various aspects was discovered to significantly enhance thefidelity of fluorescence measurements, even in the presence ofsubstantial perturbations as described herein below.

Detailed descriptions of devices for monitoring the fluorescence of anexogenous fluorescent agent in vivo and methods of correcting thefluorescence measurements to remove the effects of the diffusereflectance of light within the tissue of the patient are providedherein below.

Although the devices and methods are described herein below in thecontext of a non-invasive optical renal function monitor, it is to beunderstood that the correction method described herein, with appropriatemodification, may be applied to any compatible device configured toperform measurements by delivering EM radiation from an external sourcethrough any scattering medium and/or receiving EM radiation propagatedthrough any scattering medium to an external detector. Non-limitingexamples of EM radiation include visible light, near-IR light, IR light,UV radiation, and microwave radiation. The scattering media may includeany living or non-living material capable of propagating EM radiation ofat least one EM frequency without limitation. At least a portion of thescattering media may further include one or more substructures orcompounds capable of reflecting and/or absorbing the EM radiation.Non-limiting examples of scattering media include: a tissue of a livingor dead organism, such as a skin of a mammal; a gas such as air with orwithout additional particles such as dust, fluid droplets, or a solidparticulate material; a fluid such as water with or without additionalparticles such as gas bubbles or a solid particulate material. Further,the devices and methods described herein below are not limited todetection of renal function, but may be modified for use in thedetection of the function of other physiological systems including, butnot limited to, liver systems, or gastro-intestinal systems.

System Description

In various aspects, the methods of correcting fluorescence measurementsto remove the effects of variations in local skin properties may beincorporated into any fluorescence monitoring system including, but notlimited to, a system for optically monitoring renal function in vivo andin real time by measuring changes in fluorescence of an exogenousfluorescent agent injected into a patient as the agent is renallyeliminated from the patient. FIG. 2 is a block diagram of a system 200for optically monitoring renal function of a patient 202 viameasurements of the fluorescence of an injected exogenous fluorescentagent in the patient 202, in one aspect. The system 200 may include atleast one sensor head 204 configured to deliver light at an excitatorywavelength (λ_(ex)) into a first region 206 of the patient 202. Thesystem 200 is further configured to detect light at an emissionwavelength (λ_(em)), at a second region 208 of the patient 202, and todetect light at the excitatory wavelength (λ_(ex)), and/or emissionwavelength (λ_(em)), at a third region 210 of the patient 202.

The system 200 may further include a controller 212 operatively coupledto the at least one sensor head 204, an operation unit 214, and adisplay unit 216. In various aspects, the controller 212 is configuredto control the operation of the at least one sensor head 204 asdescribed in additional detail herein below. The controller 212 isfurther configured to receive measurements of light from the at leastone sensor head 204. The controller 212 is further configured to correctthe light measurements corresponding to fluorescence from exogenousfluorescent agents according to at least one method including, but notlimited to, the disclosed methods of correcting fluorescencemeasurements using measurements of the diffuse reflectance of light. Thecontroller 212 is further configured to transform the fluorescencemeasurements received from the at least one sensor head 204 into asummary parameter representative of the renal function of the patient202. In addition, the controller 212 is configured to receive at leastone signal representing user inputs from the operation unit 214 and togenerate one or more forms for display on the display unit 216including, but not limited to, a graphical user interface (GUI).

A detailed description of the sensor head 204 and controller 212 areprovided herein below.

A. Sensor Head

In various aspects, the sensor head 204 includes at least one lightsource and at least one light detector in a housing. FIG. 6 is a sideview of a housing 600 for the sensor head 204 in one aspect thatincludes an upper housing 602 and a lower housing 604 attached togetherto enclose two light sources and two light detectors. The bottom surface608 of the lower housing 604 further includes a contact surface 606configured to be attached to the skin of a patient 202 using abiocompatible adhesive material including, but not limited to, asurgical adhesive. In use, the surface of the adhesive material oppositeto the contact surface 606 may be affixed to the skin of the patient202. In various aspects, the adhesive material may be configured totransmit light through the light sources into the patient and to furthertransmit the fluorescence from the patient to the light detectors. Inone aspect, the adhesive material may be an optically transparentmaterial. In another aspect, the adhesive material may be produced froma non-fluorescing material to prevent the production of confoundingfluorescence by the adhesive material.

In various other aspects, the upper housing 602 may further include oneor more openings 806 configured to provide access to the interior for acable including, but not limited to, a USB cable, and/or to provide awindow for a display generated by the circuitry contained within thehousing 600, such as an indicator LED.

FIG. 7 is a bottom view of the housing 600 illustrated in FIG. 8. Thecontact surface 606 may include an aperture plate 702 including one ormore apertures 704 configured to transmit light between the skin of thepatient and the light sources and light detectors contained inside thehousing 600. In one aspect, the aperture plate 702 may be epoxied intothe lower housing 604 to prevent liquid ingress into the interior of thehousing 600. In various aspects, the dimensions, arrangement, and/orspacing of the one or more apertures 704 may be selected to enhancevarious aspects of the operation of the system 200, as described inadditional detail herein below. In another aspect, the contact surface606 may further include a temperature sensor opening 706 configured toprovide a thermal path from the skin surface of the patient to anadditional temperature sensor 228 configured to monitor the temperatureat the skin surface of the patient.

FIG. 8 is a schematic diagram illustrating the arrangement of theelectrical components within the housing 600. Referring to FIG. 8, theupper housing 602 and the lower housing 604 may be affixed together withscrews 802, and the screw holes and the interface between the twohousing pieces may be filled with a water-resistant filler material 804including, but not limited to, a silicone material such as roomtemperature vulcanization silicone (RTV) to inhibit liquid ingress intothe interior of the housing 600.

In an aspect, the housing 600 may further include a cable opening 806formed through the upper housing 602. The cable opening 806 may beconfigured to provide access to the interior for an electrical cableincluding, but not limited to, a USB cable. In one aspect, the cable mayenable the supply of power to the light sources, light detectors,indicator lights, and associated electrical devices and circuits asdescribed herein below. In another aspect, the cable may further enablethe communication of control signals into the housing to enable theoperation of the electrical components within the housing 600, and thecable may further enable the communication of data signals encodingmeasurements obtained by one of more of the sensor devices containedwithin the housing 600 including, but not limited to: the first lightdetector 222, the second light detector 224, any additional lightdetectors, such as a first monitor photodiode 904 and a second monitordiode 906, and any additional temperature sensors 228 (see FIG. 9). Inan aspect, the cable may be attached to the cable opening 806 andadjacent upper housing 602 with a light absorbent adhesive including,but not limited to, black epoxy and may further be sealed against waterincursion using a water resistant filler material including, but notlimited to, RTV.

In an additional aspect, the housing 600 may further include at leastone display opening 808 formed through the upper housing 602. In oneaspect, each display opening 808 may be configured to provide a windowfor a display generated by the circuitry contained within the housing600, such as an indicator LED 810. In an aspect, each indicator LED 810may be positioned on a circuit board 812. In an aspect, a light pipe 814may be epoxied into the display opening 808 within the upper housing 602above each indicator LED 810. Each a light pipe 814 may be filled with awater-resistant filler material such as RTV for liquid ingressprotection. In various aspects, the at least one indicator LED 810 mayilluminate in a predetermined pattern to enable a user of the system 200to monitor the operational status of the sensor head 204.

FIG. 9 is a close-up view of the interior optical region of the sensorhead 204 showing the arrangement of the light sources 218/220 and thelight detectors 222/224 within the housing 600 in one aspect. In anaspect, the light sources 218/220 are separated from the light detectors222/224, and the first light detector 222 is separated from the secondlight detector 224 are separated from one another by a sensor mount 912affixed to the aperture plate 702. In an aspect, the sensor mount 912ensures that light from the light sources 218/220 does not reach thelight detectors 222/224 without coupling through the skin of the patient202. The separation between the first light detector 222 within thefirst detection well 908 and the second light detector 224 within thesecond detection well 910 ensures that the fluorescence signal producedby the exogenous fluorescent agent within the tissues of the patient 202is distinguishable from the unfiltered excitation light introduced bythe first light source 218.

Referring again to FIG. 9, the sensor mount 912 may be aligned to acircuit board (not shown) containing the light sources 218/220 and lightdetectors 222/224 using alignment pins 914 and held in place usingscrews 916. In an aspect, the sensor mount 912 may be affixed to thecircuit board containing the light sources 218/220 and light detectors222/224 using a light absorbent adhesive including, but not limited to,black epoxy. In this aspect, this light-resistant join between thecircuit board and the sensor mount 912 inhibits leakage of light betweenthe light sources 218/220 and the light detectors 222/224, and furtherinhibits the leakage of light between the first light detector 222 andthe second light detector 224. The apertures 704 configured to transmitlight to and from the skin underlying the contact surface 606 of thesensor head 204 are formed through a structurally separate apertureplate 702 (see FIG. 7) to provide for precise alignment of the apertures704 to the corresponding light sources 218/220 and light detectors222/224, described in additional detail herein below.

In various aspects, the sensor mount 912 may further provide electricalshielding for any sensitive electrical devices within the sensor head204 including, but not limited to, the light detectors 222/224. In oneaspect, the sensor mount 912 may be constructed of an electricallyconductive material including, but not limited to: aluminum and aluminumalloy. In this aspect, the sensor mount 912 may be electrically coupledto the ground of the circuit board using conductive screws 916. Inaddition, any glass windows positioned within the source well 902 and/ordetector wells 908/910 adjacent to the aperture plate 702 including, butnot limited to, an optical filter 244 and clear glass 246 as describedherein below (see FIG. 2) may further include an electrically conductivecoating. Non-limiting examples of suitable electrically conductivecoatings for the glass windows of the sensor mount include a conductiveindium tin oxide (ITO) coating and any other suitable transparent andelectrically conductive coating.

Without being limited to any particular theory, the conductive materialof the sensor mount 912 provides a partial Faraday cage to shield theelectrically sensitive detectors 222/224 from electrical noise generatedby or conducted through the patient's body. The partial Faraday cageprovided by the sensor mount 912 may be completed with the conductiveITO coating on the glass windows within the source well 902 and/ordetector wells 908/910. In an aspect, the electrically conductivecoating on the glass windows, such as an ITO coating, are sufficientlyconductive to provide electrical shielding while remaining sufficientlytransparent for the transmission of light to and from the skin surfaceof the patient 202. In another aspect, the ITO coating of each glasswindow may be grounded to an electrically conductive sensor mount 912using any known electrical grounding method including, by not limitedto: a wire connecting the glass coating to the sensor mount 912 that isattached at both wire ends with conductive epoxy, or attaching thecoated glass directly to a glass fitting such as a ledge or frame formedwithin each of the source well 902 and/or detector wells 908/910 usingan electrically conductive epoxy.

In various aspects, the contact surface 606 of the housing 600 may beattached the patient's skin using a biocompatible and an adhesivematerial 610 including, but not limited to, a clear double-sided medicalgrade adhesive, as illustrated in FIG. 6 and FIG. 7. Any adhesivematerial selected to be optically transmissive at the excitation andemission wavelengths used by the system 100 as described herein. Theadhesive material 610 may be positioned on the contact surface 606 suchthat the adhesive material covers the apertures 704, but exposes thetemperature sensor opening 706 to ensure sufficient thermal contact withthe skin of the patient 202. In one aspect, the sensor head 204 may befurther secured to the patient 202 as needed using one or moreadditional biocompatible medical fastener devices including, but notlimited to: Tegaderm bandages, medical tape, or any other suitablebiocompatible medical fastener devices.

In an aspect, the contact surface 606 may be located near the front edgeof the sensor head 204 to provide for accurate positioning of thecontact surface 606 on a selected region of the patient's skin. Inanother aspect, the apertures 704 may be positioned towards the centerof the contact surface 606 to reduce ambient light ingress. Withoutbeing limited to any particular theory, ambient light may enter one ormore of the apertures 704 due to incomplete adhesion of the contactsurface 606 to the patient's skin and/or due to the propagation ofambient light passing through the patient's exposed skin situated justoutside of the footprint of the contact surface 606 into the apertures704.

Referring again to FIG. 6, the bottom surface 608 of the sensor head 204curves away from the plane of the contact surface 606 to enableattachment of the sensor head 204 to varied body type and locations. Forattachment of the sensor head 204 to relatively flat or concavesurfaces, any gap 612 between the bottom surface 608 and the skinsurface of the patient 202 may be filled with a biocompatible foam toensure consistent contact with the patient 202.

i) Light Sources

In various aspects, each sensor head 204 includes a first light source218 and a second light source 220 configured to deliver light to a firstregion 206 of a patient 202. The first light source 218 is configured todeliver the light at the excitatory wavelength and the second lightsource 220 is configured to deliver light at the emission wavelength. Inone aspect, the excitatory wavelength may be selected to fall within aspectral range at which the exogenous fluorescent agent exhibitsrelatively high absorbance. In another aspect, the emission wavelengthmay be selected to fall within a spectral range at which the exogenousfluorescent agent exhibits relatively high emission. The exogenousfluorescent agent may be selected for enhanced contrast relative toother chromophores within the tissues of the patient 202 including, butnot limited to hemoglobin within red blood cells and/or melanin withinmelanocytes. In various aspects, the exogenous fluorescent agent may beselected to conduct measurements within spectral ranges with lowervariation in absorption by other chromophores such as hemoglobin withinthe tissues of the patient 202 during use.

Without being limited to any particular theory, hemoglobin (Hb) is anabsorber of visible light in the tissues of the patient 202, and has thepotential to interfere with the measurements of fluorescence of theexogenous fluorescent agent if the Hb absorbance varies over themeasurement period of the system 200. Because hemoglobin (Hb) enablesgas exchange within virtually all tissues containing circulatoryvessels, virtually all tissues are vulnerable to interference withfluorescence measurements of the system 200 due to fluctuations inhemoglobin concentration. Within most tissues, externally appliedpressure may cause blood pooling which may be manifested as an apparentdecay of the fluorescence measured at the skin surface. Periodic openingand closing of blood vessels (“vasomotion”) near the surface of the skinmay also cause fluctuations in hemoglobin concentration which mayintroduce additional noise in to measurements of fluorescence of theexogenous fluorescent agent by the system 200. Further, in some patients202, such as those with pulmonary disorders, variation in the Hboxygenation state may also be observed, leading to additional potentialvariations in the background skin absorbance due to differences in theabsorption spectra of deoxyhemoglobin (Hb) and oxyhemoglobin (HbO₂),shown illustrated in FIG. 3.

In an aspect, the excitation and emission wavelengths for the exogenousfluorescent agent may be selected to coincide with a pair of HbO₂/Hbisosbestic points, each isosbestic point defined herein as a wavelengthcharacterized by about equal light absorbance by HbO₂ and Hb. Withoutbeing limited to any particular theory, fluorescence measurementsconducted at each isosbestic wavelength are less sensitive to variationdue to changes in the oxygenation of hemoglobin, so long as the combinedconcentration of HbO₂ and Hb remains relatively stable duringmeasurements of fluorescence by the system 200. Non-limiting examples ofHb/HbO₂ isosbestic wavelengths include: about 390 nm, about 422 nm,about 452 nm, about 500 nm, about 530 nm, about 538 nm, about 545 nm,about 570 nm, about 584 nm, about 617 nm, about 621 nm, about 653 nm,and about 805 nm.

In various aspects, the excitation and emission wavelengths may beselected based on the absorption and emission wavelengths of theselected exogenous fluorescent agent of the system 200. In one aspect,the excitatory wavelength may be an HbO₂/Hb isosbestic wavelength andsimultaneously may be a wavelength within a spectral range of highabsorbance of the exogenous fluorescent agent. In another aspect, theemission wavelength may be an HbO₂/Hb isosbestic wavelength andsimultaneously may be a wavelength within a spectral range of emissionby the exogenous fluorescent agent. Table 3 provides a summary ofHbO2/Hb isosbestic wavelengths within the spectral range of 200 nm toabout 1000 nm. FIG. 4 is a graph of the absorption spectra used toidentify the HbO₂/Hb isosbestic wavelengths of Table 1.

TABLE 1 HbO₂/Hb Isosbestic Wavelengths λ = 200-1000 nm Excitation HbMolar Wavelength Extinct. Coeff. HbO₂ dA/dλ Hb dA/dλ (nm) (M⁻¹ cm⁻¹)(M⁻¹ cm⁻¹ nm⁻¹) (M⁻¹ cm⁻¹ nm⁻¹) 260 1.2 × 10⁵ 1.8 × 10³ 6.3 × 10² 2881.1 × 10⁵ −2.9 × 10³  −3.4 × 10³  298 7.0 × 10⁴ −3.3 × 10³  −3.2 × 10³ 314 6.5 × 10⁴ 1.6 × 10³ 1.5 × 10³ 324 8.2 × 10⁴ 1.9 × 10³ 1.8 × 10³ 3401.1 × 10⁵ 6.5 × 10² 1.6 × 10³ 390 1.7 × 10⁵ 1.0 × 10⁴ 5.1 × 10³ 422 4.3× 10⁵ −2.6 × 10⁴  1.3 × 10⁴ 452 6.3 × 10⁴ −2.3 × 10³  −1.7 × 10⁴  5002.1 × 10⁴ −1.7 × 10²  4.8 × 10² 530 3.9 × 10⁴ 2.0 × 10³ 7.2 × 10² 5455.1 × 10⁴ −1.3 × 10³  7.0 × 10² 570 4.5 × 10⁴ 2.2 × 10³ −9.0 × 10²  5843.4 × 10⁴ −4.1 × 10³  −7.1 × 10²  738 1.1 × 10³ 6.8 × 10⁰ 3.5 × 10⁰ 7968.0 × 10² 8.8 × 10⁰ 1.1 × 10¹

By way of illustrative example, FIG. 3 is a graph summarizing theabsorption spectra for HbO₂ and Hb, as well as the absorption andemission spectra of frequency spectra of MB-102, an exogenousfluorescent agent in one aspect. Emission spectra for a blue LED lightsource and a green LED light source are also shown superimposed over theother spectra of FIG. 3. In this aspect, the system 200 may include ablue LED as the first light source 218, and the excitatory wavelengthfor the system 200 may be the isosbestic wavelength of about 450 nm. Aslisted in Table 1 and shown in FIG. 3, the Hb absorbance spectra isstrongly sloped at the isosbestic wavelengths of about 420 nm to about450 nm (see columns 3 and 4 of Table 1), indicating that the relativeabsorbance of HbO₂ and Hb at the isosbestic wavelength of about 450 nmis sensitive to small changes in excitatory wavelength. However, atwavelengths above about 500 nm, the HbO₂/Hb spectra are less steeplysloped, and a broader band light source including, but not limited to,an LED with a bandpass filter may suffice for use as a first lightsource 218.

In another aspect, the excitatory wave length may be selected to enhancethe contrast in light absorbance between the exogenous fluorescent agentand the chromophores within the tissues of the patient 202. By way ofnon-limiting example, as shown in FIG. 3 at the isosbestic wavelength of452 nm, the light absorption of the MB-102 is more than three-foldhigher than the light absorption of the HbO₂ and the Hb. Without beinglimited to any particular theory, a higher proportion of lightilluminating the tissue of the patient 202 at a wavelength of about 450nm will be absorbed by the MB-102 relative to the HbO₂ and Hb, thusenhancing the efficiency of absorption by the MB-102 and reducing theintensity of light at the excitatory wavelength needed to elicit adetectable fluorescence signal.

In various aspects, a second isosbestic wavelength may also be selectedas the emission wavelength for the system 200. By way of non-limitingexample, FIG. 3 shows an emission spectrum of the MB-102 exogenouscontrast agent that is characterized by an emission peak at a wavelengthof about 550 nm. In this non-limiting example, the isosbestic wavelengthof 570 nm may be selected as the emission wavelength to be detected byfirst and second detectors 222/224. In various other aspects, theemission wavelength of the system 200 may be selected to fall within aspectral range characterized by relatively low absorbance of thechromophores within the tissues of the patient 202. Without beinglimited to any particular theory, the low absorbance of the chromophoresat the selected emission wavelength may reduce the losses of lightemitted by the exogenous fluorescent agent and enhancing the efficiencyof fluorescence detection.

In various aspects, the first light source 218 and the second lightsource 220 may be any light source configured to deliver light at theexcitatory wavelength and at the emission wavelength. Typically, thefirst light source 218 delivers light at an intensity that is sufficientto penetrate the tissues of the patient 202 to the exogenous fluorescentagent with sufficient intensity remaining to induce the emission oflight at the emission wave length by the exogenous fluorescent agent.Typically, the first light source 218 delivers light at an intensitythat is sufficient to penetrate the tissues of the patient 202 to theexogenous fluorescent agent with sufficient intensity remaining afterscattering and/or absorption to induce fluorescence at the emission wavelength by the exogenous fluorescent agent. However, the intensity oflight delivered by the first light source 218 is limited to an uppervalue to prevent adverse effects such as tissue burning, cell damage,and/or photo-bleaching of the exogenous fluorescent agent and/or theendogenous chromophores in the skin (“auto-fluorescence”).

Similarly, the second light source 220 delivers light at the emissionwavelength of the exogenous fluorescent agent at an intensity configuredto provide sufficient energy to propagate with scattering and absorptionthrough the first region 206 of the patient and out the second region208 and third region 210 with sufficient remaining intensity fordetection by the first light detector 222 and the second light detector224, respectively. As with the first light source 218, the intensity oflight produced by the second light source 220 is limited to an uppervalue to prevent the adverse effects such as tissue injury orphotobleaching described previously.

In various aspects, the first light source 218 and the second lightsource 220 may be any light source suitable for use with fluorescentmedical imaging systems and devices. Non-limiting examples of suitablelight sources include: LEDs, diode lasers, pulsed lasers, continuouswaver lasers, xenon arc lamps or mercury-vapor lamps with an excitationfilter, lasers, and supercontinuum sources. In one aspect, the firstlight source 218 and/or the second light source 220 may produce light ata narrow spectral bandwidth suitable for monitoring the concentration ofthe exogenous fluorescence agent using the method described herein. Inanother aspect, the first light source 218 and the second light source220 may produce light at a relatively wide spectral bandwidth.

In one aspect, the selection of intensity of the light produced by thefirst light source 218 and the second light source 220 by the system 200may be influenced any one or more of at least several factors including,but not limited to, the maximum permissible exposure (MPE) for skinexposure to a laser beam according to applicable regulatory standardssuch as ANSI standard Z136.1. In another aspect, light intensity for thesystem 200 may be selected to reduce the likelihood of photobleaching ofthe exogenous fluorescent source and/or other chromophores within thetissues of the patient 202 including, but not limited to: collagen,keratin, elastin, hemoglobin within red blood cells and/or melaninwithin melanocytes. In yet another aspect, the light intensity for thesystem 200 may be selected in order to elicit a detectable fluorescencesignal from the exogenous fluorescent source within the tissues of thepatient 202 and the first light detector 222 and/or second lightdetector. In yet another aspect, the light intensity for the system 200may be selected to provide suitably high light energy while reducingpower consumption, inhibiting heating/overheating of the first lightsource 218 and the second light source 220, and/or reducing the exposuretime of the patient's skin to light from the first light detector 222and/or second light detector.

In various aspects, the intensity of the first light source 218 and thesecond light source 220 may be modulated to compensate any one or moreof at least several factors including, but not limited to: individualdifferences in the concentration of chromophores within the patient 202,such as variation in skin pigmentation. In various other aspects, thedetection gain of the light detectors may be modulated to similarlycompensate for variation in individual differences in skin properties.In an aspect, the variation in skin pigmentation may be between twodifferent individual patients 202, or between two different positions onthe same patient 202. In an aspect, the light modulation may compensatefor variation in the optical pathway taken by the light through thetissues of the patient 202. The optical pathway may vary due to any oneor more of at least several factors including but not limited to:variation in separation distances between the light sources and lightdetectors of the system 200; variation in the secure attachment of thesensor head 204 to the skin of the patient 202; variation in the lightoutput of the light sources due to the exposure of the light sources toenvironmental factors such as heat and moisture; variation in thesensitivity of the light detectors due to the exposure of the lightdetectors to environmental factors such as heat and moisture; modulationof the duration of illumination by the light sources, and any otherrelevant operational parameter.

In various aspects, the first light source 218 and the second lightsource 220 may be configured to modulate the intensity of the lightproduced as needed according to any one or more of the factors describedherein above. In one aspect, if the first light source 218 and thesecond light source 220 are devices configured to continuously varyoutput fluence as needed, for example LED light sources, the intensityof the light may be modulated electronically using methods including,but not limited to, modulation of the electrical potential, current,and/or power supplied to the first light source 218 and/or the secondlight source 220. In another aspect, the intensity of the light may bemodulated using optical methods including, but not limited to: partiallyor fully occluding the light leaving the first light source 218 and thesecond light source 220 using an optical device including, but notlimited to: an iris, a shutter, and/or one or more filters; divertingthe path of the light leaving the first light source 218 and the secondlight source 220 away from the first region 206 of the patient using anoptical device including, but not limited to a lenses, a mirror, and/ora prism.

In various aspects, the intensity of the light produced by the firstlight source 218 and the second light source 220 may be modulated viacontrol of the laser fluence, defined herein as the rate of energywithin the produced light beam. In one aspect, the laser fluence may belimited to ranges defined by safety standards including, but not limitedto, ANSI standards for exposure to laser energy such as ANSI Z136.1.Without being limited to any particular theory, the maximum fluence oflight delivered to a patient 202 may be influenced by a variety offactors including, but not limited to the wavelength of the deliveredlight and the duration of exposure to the light. In various aspects, themaximum fluence of light may range from about 0.003 J/cm2 for light atdelivered at wavelengths of less than about 302 nm to about 1 J/cm2 forlight delivered at wavelengths ranging from about 1500 nm to about 1800nm for a duration of up to about 10 sec. For light delivered atwavelengths ranging from about 400 nm to about 1400 nm (visible/NIRlight) the maximum fluence may be about 0.6 J/cm2 for a duration of upto about 10 sec, and up to about 0.2 J/cm2 for a duration ranging fromabout 10 sec to about 30,000 sec. For extended exposures, the deliveredlight is limited to a maximum power density (W/cm2) according to ANSIstandards: visible/NIR light is limited to 0.2 W/cm2 and far IR light islimited to about 0.1 W/cm2. Without being limited to a particulartheory, extended exposure to light delivered at UV wavelengths is nottypically recommended according to ANSI standards.

In another aspect, the fluence of light at the excitatory wavelengthproduced by the first light source 218 may be modulated in order toprovide sufficient energy to propagate through the skin in the firstregion 206 of the patient 202 to the exogenous fluorescent agent withoutphotobleaching, and to illuminate the exogenous fluorescent agent withenergy sufficient to induce detectable fluorescence at the first lightdetector 222 and/or the second light detector 224. In an additionalaspect, the fluence of light at the emission wavelength produced by thesecond light source 220 may be modulated in order to provide sufficientenergy to propagate through the skin in the first region 206 of thepatient 202 and through the skin in the second region 208 and the thirdregion 210 without photobleaching to emerge as detectable light at thefirst light detector 222 and the second light detector 224,respectively. By way of non-limiting example, the fluence of lightproduced by a light source at 450 nm or 500 nm may be limited to 1.5 and5 mW/cm², respectively, to prevent photo-bleaching.

In various aspects, the fluence of the light produced by the first lightsource 218 and the second light source 220 may be modulated by anysuitable systems and/or devices without limitation as described hereinabove. This modulation may be enabled a single time during operation ofthe system 200, and as a result, the fluence of the light produced byeach of the first light source 218 and the second light source 220 maybe relatively constant throughout the operation of the system 200. Inanother aspect, the light modulation may be enabled at discrete timesover the duration of operation of the system 200, or the lightmodulation may be enabled continuously over the duration of operation ofthe system 200.

In one aspect, the fluence of the light may be modulated via manualadjustment of any of the power source settings and/or optical devicesettings as described above when the system 200 is configured in anEngineering Mode. In another aspect, the fluence of the light may bemodulated automatically via one or more control schemes encoded in thelight source control unit of the controller 212 as described hereinbelow. In this aspect, the degree of modulation may be specified atleast in part on the basis of feedback measurements obtained by varioussensors provide in the sensor head 204 of the system 200 including, butnot limited to, additional light detectors 226 and temperature sensors228 as described in additional detail herein below.

In various aspects, light produced by the first light source 218 and thesecond light source 220 are further characterized by a pulse width,defined herein as the duration of the produced light. Although pulsewidth is typically used to characterize the performance of a lightsource that produces light in discrete pulses, such as a pulsed laser,it is to be understood that the term “light pulse”, as used herein,refers to any discrete burst of light produced by a single light sourceat a single wavelength to enable the acquisition of a single measurementof fluorescence by the system 200. Similarly, the term “pulse width”, asused herein, refers to the duration of a single light pulse produced bya single light source. The pulse width is typically selected based onone or more of at least several factors including, but not limited to:delivery of sufficient light energy to elicit detectable fluorescencefrom the exogenous fluorescent agent without photobleaching theexogenous fluorescent agent or other chromophores within the tissues ofthe patient 202; compliance with safety standards for light delivery topatients such as ANSI standards; light delivery at sufficiently highrate to enable data acquisition at a rate compatible with real-timemonitoring of renal function; performance capabilities of the selectedlight sources, light detectors, and other devices of the system 200;preservation of the working life of light sources, light detectors, andother devices related to producing and detecting light energy; and anyother relevant factors.

In various aspects, the pulse width of the light produced by the firstlight source 218 and the second light source 220 may be independentlyselected to be a duration ranging from about 0.0001 seconds to about 0.5seconds. In various other aspects, the pulse width of the light producedby the first light source 218 and the second light source 220 may beindependently selected to be a duration ranging from about 0.0001seconds to about 0.001 seconds, from about 0.0005 seconds to about 0.005seconds, from about 0.001 seconds to about 0.010 seconds, from about0.005 seconds to about 0.05 seconds, from about 0.01 seconds to about0.1 seconds, from about 0.05 seconds to about 0.15 seconds, from about0.1 seconds to about 0.2 seconds, from about 0.15 seconds to about 0.25seconds, from about 0.2 seconds to about 0.3 seconds, from about 0.25seconds to about 0.35 seconds, from about 0.3 seconds to about 0.4seconds, from about 0.35 seconds to about 0.45 seconds, and from about0.4 seconds to about 0.5 seconds. In one aspect, the pulse widths of thelight produced by the first light source 218 and the second light source220 are both about 0.1 seconds, as illustrated schematically in FIG. 5.

In another aspect, the light produced by the first light source 218 andthe second light source 220 may be further characterized by a pulserate, defined herein as the number of pulses produced by a light sourceper second. Although pulse rate is typically used to characterize theperformance of a light source that produces light in discrete pulses,such as a pulsed laser, it is to be understood that the term “pulserate”, as used herein, refers to the rate of production of a discretelight pulse by a single light source at a single wavelength inassociation with the acquisition of measurements of fluorescence by thesystem 200. In various aspects, the pulse rate may be selected based onone or more of at least several factors including, but not limited to:compliance with safety standards for light delivery to patients such asANSI standards; the performance capabilities of the selected lightsources, light detectors, and other devices of the system 200; lightdelivery rates compatible with data acquisition rates sufficiently rapidfor real-time monitoring of renal function; preserving the working lifeof light sources, light detectors, and other devices related toproducing and detecting light energy; and any other relevant factor.

In various aspects, the light sources are configured to deliver lightinto the tissues of the patient 202 at a single position such as a firstregion 206, illustrated schematically in FIG. 2. In one aspect, thedelivery of light at both the excitatory wavelength and the emissionwavelength to the same first region 206 enables both light pulses toshare at least a portion of the optical path traveled through thetissues of the patient 202 between the point of entry at the firstregion 206 and the point of detection at the second region 208 and thethird region 210. As discussed in detail herein below, this arrangementof optical paths enhances the quality of data produced by the system200.

In one aspect, the first light source 218 and the second light source220 may be operatively coupled to a common means of light delivery. Inone aspect (not illustrated) the first light source 218 and the secondlight source 220 may each be operatively coupled to a first optic fiberand a second optic fiber, respectively, and the first and second opticfibers may be joined to a third optic fiber configured to direct lightfrom the first optic fiber and/or the second optic fiber into the firstregion 206 of the patient 202. In another aspect, the first light source218 and the second light source 220 may be operatively coupled to acommon optic fiber or other optical assembly configured to direct thelight from the first light source 218 and/or the second light source 220into the first region 206 of the patient 202. In this aspect, the lightproduced by the first light source 218 and the second light source 220may be directed in an alternating pattern into the common optic fiber orother optical assembly using an adjustable optical device including, butnot limited to, dichroic mirror or a rotating mirror.

In an aspect, the system 200 may include the sensor head 204 providedwith a sensor mount 912 configured with one or more wells within whichthe light sources 218/220 and light detectors 222/224 may be attached ina predetermined arrangement. In one aspect, illustrated in FIG. 9 andFIG. 10, the first light source 218 and the second light source 220 maybe situated within a source well 902 of the sensor mount 912 positionedwithin the sensor head 204 (see FIG. 9). In an aspect, the source well902 may contain a first LED light source 218 producing light at theexcitation wavelength and a second LED light source 220 producing lightat the emission wavelength operatively coupled to a single lightdelivery aperture 1002 (see FIG. 10) formed through the aperture plate702, which ensures that both wavelengths of light (i.e. excitatory andemission) enter the skin of the patient 202 at approximately the samelocation including, but not limited to, a first region 206 asillustrated schematically in FIG. 2. In an aspect, the source well 902further contains a first monitor photodiode 904 and a second monitorphotodiode 906, which are used to correct for variations in output powerfrom the LED light sources as described in further detail herein below.

In an aspect, only a fraction of the light energy produced by the LEDlight sources is delivered to the skin of the patient 202 via the singlelight delivery aperture 1002. In one aspect, the skin of the patient 202receives about 1% of the light energy produced by the LED light sources.In various other aspects, the skin of the patient 202 receives about 2%,about 3%, about 4%, about 5%, about 7.5%, about 10%, about 20%, andabout 50% of the light energy produced by the LED light sources. Withoutbeing limited to any particular theory, the fraction of light producedby the LED light sources delivered to the skin of the patient 202 may beincreased by the incorporation of additional optical elements configuredto focus and/or direct the light from each LED light source to the lightdelivery aperture 1002. In another aspect, a diffuser may be used to mixthe output of the light sources so that the light energy is renderedhomogeneous at the surface of the skin of the patient.

ii) Light Detectors

Referring again to FIG. 2, the system 200 further includes a first lightdetector 222 and a second light detector 224 in various aspects. In anaspect, the first light detector 222 is configured to measure unfilteredlight emitted from the tissue of the patient 202 at the second region208, and the second light detector 224 is configured to measure filteredlight emitted from the tissue of the patient 202 at the third region210. In this aspect, the second light detector 224 further comprises anoptical filter 244 configured to block light at the excitationwavelength. As a result, the first light detector 222 is configured tomeasure light received at both the excitation and emission wavelengthsand the second light detector 224 is configured to detect light receivedat the emission wavelength only. Combined with the illumination of thetissues of the patient 202 with light at the excitatory wavelength onlyand at the emission wavelength only in an alternating series (see FIG.5) the measurements from the first light detector 222 and a second lightdetector 224 may be analyzed as described herein below to measure thefluorescence of an exogenous fluorescence agent and to correct thefluorescence measurements by removing the effects of the diffusereflectance of light according to the correction methods describedherein below.

In various aspects, the second region 208 and third region 210 withinthe tissues of the patient 202, from which light is detected by thefirst light detector 222 and a second light detector 224, respectively,are each separated by a nominal distance from the first region 206 towhich light produced by the first light source 218 and the second lightsource 220 is delivered. This nominal separation distance may beselected to balance two or more effects that may impact the quality ofdata detected by the light detectors. Without being limited to anyparticular theory, as the nominal separation distance increases, thetotal detected signal from the light detectors may decrease due to lightscattering along the longer optical path between light source and lightdetector. This effect may be mitigated by the choice of emissionwavelength, which may result in a less pronounced decrease in thedetected fluorescence signal (i.e. light at the emission wavelength)relative to the signals associated with detected light at the excitationwavelengths as the nominal separation distance increases. Longer nominalseparation distances result in higher sensitivity to signal changes dueto changing tissue optical properties.

In one aspect, the nominal separation distance may range from 0 mm (i.e.colocation of light sources and light detectors) to about 10 mm. Invarious other aspects, the nominal separation distance may range fromabout 1 mm to about 8 mm, from about 2 mm to about 6 mm, and from about3 mm to about 5 mm. In various additional aspects, the nominalseparation distance may be 0 mm, about 1 mm, about 2 mm, about 3 mm,about 4 mm, about 5 mm, about 6 mm, about 8 mm, and about 10 mm. In oneaspect, the nominal separation distance may be about 4 mm to balancethese competing effects of logarithmic drop-off of signal and reducedsize of the background signal relative to the signal from the exogenousfluorescent agent.

Referring again to FIG. 9, the first light detector 222 may bepositioned within a first detection well 908 of the sensor mount 912 andthe second light detector 224 may be positioned within a seconddetection well 910 of the sensor mount 912 within the sensor head 204.The first light detector 222 and the second light detector 224 mayreceive light from tissue of the patient 202 through a first detectoraperture 1004 and second detector aperture 1006, respectively. In anaspect, the first detector aperture 1004, the second detector aperture1006, and the light delivery aperture 1002 are mutually separated fromone another by the nominal separation distance disclosed herein aboveincluding, but not limited to, a nominal separation distance of 4 mm. Inan aspect, the first detection well 908, second detection well 910, andlight source well 902 of the sensor mount 912 may be optically isolatedfrom one another to ensure that light from the light sources 218/220does not reach the light detectors 222/224 without coupling through theskin of the patient 202. The separation between the two detection wells908/910 ensures that the detected fluorescence signal from the exogenousfluorescent agent is distinguishable from the unfiltered excitationlight, as described in detail herein below.

In an aspect, the three apertures 704 of the aperture plate 702 (seeFIG. 7) are circular with a diameter ranging from about 0.5 mm to about5 mm. In various other aspects, the diameters of the apertures may rangefrom about 0.5 mm to about 1.5 mm, about 1 mm to about 2 mm, about 1.5mm to about 2.5 mm, about 2 mm to about 3 mm, about 2.5 mm to about 3.5mm, about 3 mm to about 4 mm, about 3.5 mm to about 4.5 mm, and about 4mm to about 5 mm.

In one aspect, the three apertures 704 of the aperture plate 702 arecircular apertures with a diameter of about 1 mm diameter. This finitewidth of the apertures may result in an effective source-detectorseparation of less than the nominal separation distance because of thelogarithmic drop-off of signal with increasing separation distance fromthe light sources at the skin interface of the sensor head 204.

In various aspects, the light detectors 222/224 of the system 200 may beany suitable light detection device without limitation. Non-limitingexamples of suitable light detection devices include: photoemissiondetectors such as photomultiplier tubes, phototubes, and microchannelplate detectors; photoelectric detectors such as LEDs reverse-biased toact as photodiodes, photoresistors, photodiodes, phototransistors; andany other suitable light detection devices. In an aspect, the lightdetectors 222/224 are sufficiently sensitive to detect the fluorescenceemitted by the exogenous fluorescent agents within the tissues ofpatients 202 that include melanin ranging from about 1% to about 40%melanin in the epidermis and blood volume ranging from about 0.5% toabout 2% of the skin volume. In one aspect, the light detectors 222/224may be silicon photomultiplier (SPM) devices.

In an aspect, the first light detector 222 may be configured to detectlight at both the excitatory frequency and at the emission frequency,and the second light detector 224 may be configured to detect light atthe emission frequency only. In one aspect, the second light detector224 may respond only to light of the emission wavelength as a result ofthe design and materials of the sensor elements of the second lightdetector 224. In another aspect, the second light detector 224 mayrespond to a wider range of light wavelengths, but may be positioneddownstream from an optical filter configured to pass only the portion ofincoming light with the emission wavelength and further configured toblock the passage of light wavelengths outside of the emissionwavelength.

Any suitable optical filter may be selected for use with the secondlight detector 224 to detect light selectively at the emissionwavelength. Non-limiting examples of suitable optical filters includeabsorptive filters and interference/dichroic filters. Without beinglimited to any particular theory, the performance of an absorptionfilter does not vary significantly with the angle of incident light,whereas the performance of an interference/dichroic filter is sensitiveto the angle of incident light and may require additional collimationoptics to effectively filter the Lambertian light distributionrepresentative of light emitted from the skin of the patient 202.

In one aspect, the second light detector 224 may be positioneddownstream of an absorptive long-pass filter configured to pass lightabove a predetermined wavelength to the second light detector 224. Byway of non-limiting example, the second light detector 224 may bepositioned downstream of an long-pass OG530 filter configured to passlight with wavelengths above about 530 nm. Other non-limiting examplesof suitable filters include a Hoya O54 filter and a Hoya CM500 filter.

In various aspects, an absorption filter 244 configured to absorbexcitation wavelength light may be positioned within the seconddetection well 910 between the second light detector 224 and the seconddetector aperture 1006. In one aspect, the absorption filter 244 may beconstructed from OG530 Schott glass. The thickness of the absorptionfilter 244 may be selected to enable an optical density sufficient tofilter the excitation light by about three orders of magnitude. In oneaspect, the thickness of the absorption filter 244 may range from about1 mm to about 10 mm. In various other aspects, the thickness of theabsorption filter 244 may range from about 1 mm to about 8 mm, fromabout 2 mm to about 6 mm, and from about 3 mm to about 5 mm. In variousadditional aspects, the thickness of the absorption filter 244 may beabout 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm,about 7 mm, about 8 mm, about 9 mm, and about 10 mm. In one aspect, theabsorption filter 244 is a 3-mm thick filter constructed of OG530 Schottglass.

In an additional aspect, an optical diffuser may be provided within thelight source well 902. In this aspect, the optical diffuser enablesmixing of the light entering the light source well 902 from the firstand second light sources 218/220. By mixing the light from the first andsecond light sources 218/220 using the optical diffuser prior toillumination of the first region 206 of the patient 202, the similarityof the optical paths taken by emission-wavelength light andexcitation-wavelength light through the tissues of the patient isenhanced relative to the corresponding optical paths taken by unmixedlight, thereby reducing a potential source of variation.

In an aspect, a transparent material configured to pass light of boththe excitatory and emission wavelengths may be positioned within thefirst detection well 908 between the first light detector 222 and thefirst detector aperture 1004. In this aspect, the transparent materialmay be any material with similar optical properties to the material ofthe absorption filter 244 including, but not limited to, thickness andindex of refraction. In one aspect, the transparent material within thefirst detection well 908 may be fused silica glass of the same thicknessas the absorption filter 244.

By way of non-limiting example, the transmission spectrum of the OG 530filter is provided in FIG. 3. As illustrated in FIG. 3, the transmissionspectrum of the OG 530 filter overlaps with the emission spectrum of theMB-102 exogenous fluorescent agent and the emission spectrum of a greenLED used as a second light source 220 (emission wavelength). Inaddition, the transmission spectrum of the OG 530 filter excludes theemission spectrum of the blue LED used as a first light source 218 andthe absorbance spectrum of the MB-102 exogenous fluorescent agent(excitation wavelength).

In an aspect, the transparent material such as glass 246 and theabsorption filter 244 may be secured to ledges formed within the firstdetection well 908 and the second detection well 910, respectively. Thetransparent material such as glass 246 and the optical filter 244 may besecured in place using an opaque and/or light absorbing adhesiveincluding, but not limited to, black epoxy to ensure that all lightreceived through the first detector aperture 1004 and the seconddetector aperture 1006 travels through the optical filter 244 or glass246 before detection by the first and second light detectors 222/224. Inanother aspect, the sides of the optical filter 244 or glass 246 may bepainted black with a light-absorbing coating including, but not limitedto, India ink to ensure that light does not reach the first and secondlight detectors 222/224 without passing through the optical filter 244or glass 246.

In an aspect, the height of the detection wells 908/910, combined withthe diameter of the detector apertures 1004/1006 may limit the fractionof the light emitted from the second region 208 and third region 210 ofthe patient's skin that reaches the active areas of the light detectors222/224 due to the Lambertian distribution of the angle of the lightleaving the patient's skin. In one aspect, the fraction of light emittedfrom the second region 208 and third region 210 of the patient's skinreceived by the light detectors 222/224 may range from about 5% to about90%. In various other aspects, the fraction of light may range fromabout 5% to about 15%, from about 10% to about 20%, from about 15% toabout 25%, from about 20% to about 30%, from about 25% to about 35%,from about 30% to about 40%, from about 35% to about 45%, from about 40%to about 60%, from about 50% to about 70%, and from about 60% to about90%.

In one aspect, for the sensor head 204 illustrated in FIG. 6 and FIG. 7with 1-mm diameter apertures 1002/1004/1006, about 10% of the lightemitted from the surface of the patient's skin may reach the active areaof the light detectors 222/224 to be detected. In various aspects, thesensor head 204 may further include additional optical elementsincluding, but not limited to, lenses and/or prisms configured tocompensate for the Lambertian distribution of light angles in order toenhance the fraction of light emitted from the patient's skin that isdirected to the active area of the light detectors 222/224.

iii) Temperature Sensors

Referring to FIG. 2, the sensor head 204 may further include one or moreadditional temperature sensors 228 configured to monitor temperatures ofvarious regions within the sensor head 204 and in the vicinity of thesensor head 204. Non-limiting examples of suitable regions for which thetemperature may be monitored by the one or more additional temperaturesensors 228 include: temperature at the skin surface of the patient 202;temperature in the vicinity of the first light source 218 and/or secondlight source 220; ambient temperature outside of the sensor head 204;temperature of housing 600 of sensor head 204; and any other suitableregion. In one aspect, additional temperature sensors 228 may beconfigured to monitor the temperatures in the vicinity oftemperature-sensitive electrical components including, but not limitedto: light sources 218/220 such as LEDs, light detectors 222/224 such assilicon photomultipliers (SPMs), and any other temperature-sensitiveelectrical components of the sensor head 204. In some aspects, one ormore temperatures measured by one or more additional temperature sensors228 may be used as feedbacks in a control method for one or more of thetemperature-sensitive devices of the system 200 as described hereinbelow.

By way of non-limiting example, a temperature measurement may be used tocontrol the amount of light energy produced by an LED used as a first orsecond light source 218/220. In this example, LED temperatures measuredby an second temperature sensor 1108 (see FIG. 11) may be used in acontrol scheme to modulate the amount of power supplied to an LED lightsource to compensate for the effect of LED temperature on the lightoutput of the LED. In another aspect, additional temperature sensors 228may monitor the temperatures of LED light sources 218/220 to monitorand/or compensate for temperature variations of the LEDs as well as tomonitor and/or compensate for temperature-dependent transmission of theoptical filters to maintain relatively constant output wavelengths.

By way of another non-limiting example, an additional temperature sensor228 may be included in the sensor head 204 in the form of a thermistor816 (see FIG. 8) configured to monitor the temperature of the housing600 in the vicinity of the contact surface 606 of the sensor head 204.Referring to FIG. 7, FIG. 8, and FIG. 9, the thermistor 816 may beepoxied into the temperature sensor opening 706 in the aperture plate702 in one aspect. In this aspect, the space 918 between the circuitboard (not shown) and the lower housing 604 may be filled with athermally conductive putty to ensure good thermal conduction anddissipation.

In this example, the measured housing temperature may be used tomodulate the light output of the sensor head 204 to prevent overheatingof the skin of the patient 202 during use. In another aspect, additionaltemperature sensors 228 may monitor the temperatures of LED lightsources 218/220 to monitor and/or compensate for temperature variationsof the LEDs to enable the maintenance of relatively constant outputwavelengths by the LED light sources 218/220.

In an additional aspect, temperatures measured by one or more additionaltemperature sensors 228 may provide for subject safety by disabling oneor more electrical devices including the light sources 218/220 and/orlight detectors 222/224 if an over-temperature condition is detected. Inone aspect, an over-temperature condition may be indicated if the casetemperature detected by the thermistor 816 is greater than about 40° C.In various other aspects, an over-temperature condition may be detectedof the case temperature is greater than about 40.5° C. or greater thanabout 41.0° C.

B. Controller

Referring again to FIG. 2, the system 200 in various aspects may includea controller 212 configured to operate the light sources 218/200 andlight detectors 222/224 in a coordinated fashion to obtain a pluralityof measurements used to obtain the fluorescence of the exogenousfluorescent agent within the tissues of the patient 202, to correct thefluorescence data to remove the effects of the diffuse reflectance oflight as described herein below, and to transform the fluorescencemeasurements into a parameter representative of the renal function ofthe patient 202. FIG. 11 is a schematic diagram of an electronic circuit1100 that illustrates the arrangement of various electrical componentsthat enable the operation of the system 200 in an aspect. In one aspect,the controller 212 may be a computing device further including anoperation unit 214 and a display unit 216.

i) Light Source Control Unit

Referring again to FIG. 2, the controller 212 may include a light sourcecontrol unit 230 configured to operate the first light source 218 andthe second light source 220 to produce light at the excitationwavelength and emission wavelength, respectively in a coordinated mannerto produce a repeating pulse sequence as illustrated schematically inFIG. 5. In various aspects, the light source control unit 230 mayproduce a plurality of light control signals encoding one or more lightcontrol parameters including, but not limited to: activation ordeactivation of each light source; relative timing of activation anddeactivation of each light source to enable light pulse width, pulserepetition rate, electrical power delivered to the light source or otherparameter associated with light pulse fluence or light pulse power;other light source-specific parameters controlling the light output ofthe light source; and any other relevant light control parameter. In anaspect, the light source control unit 230 may receive one or morefeedback measurements used to modulate the plurality of control signalsto compensate for variations in performance of the light sources inorder to maintain a relatively stable output of light from the lightsources. Non-limiting examples of feedback measurements used by thelight source control unit 230 include: light output of the light sources218/220 measured within the source well 902 by the first monitorphotodiode 904 and the second monitor photodiode 906, respectively,temperatures of the light sources 218/220, and any other feedbackmeasurement relevant to monitoring the performance of light sources218/220.

By way of non-limiting example, the light source control unit 230 may beconfigured to operate LED light sources 218/220. In this example, thelight output of the LED light sources 218/220 may be controlled bycontrolling the magnitude of current provided to each LED. In an aspect,the light source control unit 230 may include at least one waveformgenerator 1122 including, but not limited to, a field programmable gatearray FPGA with a 16-bit DAC 1124 operatively coupled to a LED currentsource 1126, as illustrated in FIG. 11. In an aspect, waveformsgenerated by the at least one waveform generator 1122 including, but notlimited to square waves, may control the output from the LED currentsource 1126. In an aspect, the magnitude of the current supplied to theLED light sources 218/220 may be adjustable based on the waveformsignals provided by the waveform generator/FPGA 1122.

Referring to FIG. 5, in one aspect, each light pulse sequence 500includes an emission wavelength light pulse 502 and an excitatorywavelength light pulse 504 that are both made up of a plurality ofsquare waves 506 produced by the first and second LED light sources218/220. Referring to FIG. 11, square waves generated by the waveformgenerator 1122 are received by the LED current source 1126. The currentgenerated by the LED current source includes a square waveform similarto the waveform generated by the waveform generator 1122. Without beinglimited to any particular theory, because the intensity of lightproduced by the LED light sources 218/220 is proportional to themagnitude of the current received, the light produced by the LED lightsources 218/220 also includes the square waveform as illustrated in FIG.5. In another aspect, discussed in additional detail below, the squarewaves produced by the waveform generator 1122 may also be used by theacquisition unit 234 in a synchronous detection method to reduce theeffects of various confounding factors including, but not limited to,the detection of ambient light, from the detector signals generated bythe light detectors 222/224 during illumination of the tissues of thepatient at the emission and excitatory wavelengths by the first andsecond light sources 218/220, respectively.

In various other aspects, a variety of alternate LED pulse modulationschemes may be equivalently employed without limitation. In one aspect,the excitation and emission pulses are delivered in an alternatingseries interspersed with a dark period after each pulse. In anotheraspect, the first and second LED light sources 218/220 are eachmodulated with a 50% duty cycle but at different modulation frequencies,allowing the signals associated with the excitation and emission pulsesto be separated by frequency filtering.

Without being limited to any particular theory, the overall opticalpower delivered to the patient's skin may be limited by at least twofactors: photobleaching of the exogenous fluorescent agent and/orendogenous chromophores, as well as overheating of the patient's tissuesilluminated by the system 200. In one aspect, tissue heating may imposean absolute limit of about 9 mW on the optical power that can bedelivered to the skin, based on safety standards including, but notlimited to, ANSI/IESNA RP-27.1-05. In another aspect, photobleaching ofthe skin autofluorescence associated with endogenous chromophoresincluding, but not limited to, collagen, hemoglobin, and melanin maycontribute a background signal to the measured fluorescence that remainsrelatively constant so long as no autobleaching of the chromophoresoccurs. This constant autofluorescence background may be subtracted fromthe raw fluorescence signal, but if autofluorescence varies over timedue to photobleaching, this background correction may interfere with thekinetic calculation of the renal decay time constant (RDTC). In anaspect, the light output power of the first light source 218 and/orsecond light source 220 may be limited to levels below power thresholdsassociated with chromophore photobleaching.

Referring again to FIG. 9, the light output of the light sources 218/220may be measured using monitor photodiodes 904/906 in various aspects.Because the light intensity reaching these monitor photodiodes 904/906is typically much stronger than the light intensity that reaches thelight detectors 222/224 through the patient's skin, less sensitive lightdetecting devices including, but not limited to, PIN photodiodes may beused to monitor the output of the light sources 218/220.

In various aspects, the system 200 may be configured to operate over arange of skin tones observed in the human population. Without beinglimited to any particular theory, variations in skin tones betweendifferent patients 202 may result in variations in the detectedfluorescence signals ranging over about three orders of magnitude. Inaddition, variations in the concentrations of exogenous fluorescentagent within each patient 202 may vary over a range of about two ordersof magnitude due to renal elimination of the agent over time. In variousaspects, the system 200 may be configured to detect fluorescence fromthe endogenous fluorescent agent over an intensity range of more thanfive orders of magnitude. In these various aspects, the system 200 maybe configured by modulation of at least one operational parameterincluding, but not limited to: magnitude of light output by the lightsources 218/220 and sensitivity of light detectors 222/224 correspondingto detector gains.

In one aspect, the intensity of the light output by the light sources218/220 may be manually set by a user via the operation unit 214. Inanother aspect, the light source control unit 230 may be configured tomodulate the intensity of light produced by the light sources 218/220automatically. In an aspect, the light source control unit 230 may beconfigured to control the light intensity produced by the LED lightsources 218/220 within a range of normalized output intensities from 0(off) to 1 (maximum power). In an aspect, the intensity of the lightsources 218/220 may be set by the light source control unit 230 incoordination with the detector gains of the light detectors 222/224 setby the light detector control unit 232, as described herein below.

In one aspect, signals obtained during the first 10 detection cyclesobtained by the system 200 after initialization of data acquisition, butprior to the injection of the exogenous fluorescent agent, may be usedby the light source control unit 230 to automatically adjust the lightintensity produced by the LED light sources 218/220, as well as the gainof the light detectors 222/224. In this example, the initial detectioncycle may be obtained with the LED light sources 218/220 set at about10% of maximum LED intensity (corresponding to a normalized outputintensity of 0.1) and with a low gain setting for the light detectors222/224. Based on the detected intensity of light received at the lightdetectors 222/224 at the excitation and emission wavelengths for onedetection cycle, the corresponding LED intensities may be modulated toenable the analog signals produced by the light detectors 222/224 tocorrespond to about ¼ of the full range of each detectoranalog-to-digital convertor (ADC) at the low detector gain setting. Ifthe signals produced by the light detectors 222/224 in response to thelight produced by the second LED light source 220 at the emissionwavelength do not agree, the larger signal may be used to modulate thepower setting of the second LED light source 220. If the methoddescribed above results in modulation to an LED intensity setting higherthan the maximum intensity (corresponding to a normalized outputintensity of 0.1), the LED intensity setting is set to the maximumsetting. Without being limited to any particular theory, the targetedlevels of signals produced by the light detectors 222/224 (i.e. ¼ of theADC range) is selected to reserve additional light detection capacity todetect signals resulting from variations in optical properties of thetissues of the patient 202 during the study due to any one or more of aplurality of factors including, but not limited to, the introduction ofthe exogenous fluorescent agent into the patient 202.

In the above one aspect, once the LED intensities are set by the lightsource control unit 230 in coordination with the detector gains of thelight detectors 222/224 set by the light detector control unit 232 overthe first 10 detection cycles, an additional 10 detection cycles areobtained to confirm the suitability of these settings for operation ofthe system 200 given the tissue properties of the particular patient202, followed by a recalculation of the LED intensity settings anddetector gains as described herein. If the newly calculated LEDintensity is within a factor of two of the previously determinedsetting, and the detector gains are not changed, the previouslydetermined settings are maintained for subsequent data acquisitioncycles used to determine renal function. Otherwise, the settings areupdated using the same method described herein and another 10 dataacquisition cycles conducted to confirm the stability of the settings.This process repeats until either the settings are determined to beacceptably stable or 10 data acquisition cycles are conducted to obtainthe settings, in which case the most recently determined settings areused for all subsequent data acquisitions, and the user may be notifiedvia the display unit 216 that the settings may not be optimal.

ii) Light Detector Control Unit

Referring again to FIG. 2, the controller 212 may include a lightdetector control unit 232 configured to operate the first light detector222 and the second light detector 224 to enable the detection of lightat the emission wavelength and unfiltered light at all wavelengths,respectively. In various aspects, the light detector control unit 232may produce a plurality of detector control signals encoding one or moredetector control parameters including, but not limited to, detectorgains. In various other aspects, the light detector control unit 232 mayproduce a plurality of light measurement signals encoding the intensityof light detected by the light detectors 222/224 including, but notlimited to raw detector signals that may be received by ananalog-to-digital convertor (ADC) 1102 (see FIG. 11) in various aspects.In another aspect, the detector gains and/or other detector controlsignals may be manually set by a user detector gains when the system 200is configured in an Engineering Mode.

In various other aspects, the amount of light received by the lightdetectors 222/224 may vary due to any one or more of at least severalfactors including, but not limited to: variation in skin tones observedbetween individual patients 202, variations in the concentrations ofexogenous fluorescent agent within each patient 202, and any otherrelevant parameter. In one aspect, gains of the first light detector 222and the second light detector 224 may be set by a user via the operationunit 214. In another aspect, the light detector control unit 232 may beconfigured to modulate the gain of the light detectors 222/224automatically via a bias voltage gain of the bias voltage generator 1112(see FIG. 11).

In one aspect, signals obtained during the first 10 detection cyclesobtained by the system 200 after initialization of data acquisition, butprior to the injection of the exogenous fluorescent agent, may be usedby the light detector control unit 232 to automatically adjust the gainsof the light detectors 222/224, as well as the output intensities of thelight sources 218/220. As described herein previously, the initialdetection cycle may be obtained with the LED light sources 218/220 setat about 10% of maximum LED intensity (corresponding to a normalizedoutput intensity of 0.1) and with a low gain setting for the lightdetectors 222/224 and the LED intensities may be modulated to enable theanalog signals produced by the light detectors 222/224 to correspond toabout ¼ of the full range of each detector analog-to-digital convertor(ADC) at the low detector gain setting.

In this one aspect, if the intensity of the first LED light source 218(producing light at the excitation wavelength) is set to the maximum ofthe LED power range, a high detector gain may be considered for thesecond light detector 224 corresponding to the filtered measurements ofthe excitation wavelength only. In various aspects, the high detectorgain may be 10-fold higher than the corresponding low detector gain fora given light detector. Without being limited to any particular theory,the expected peak detected fluorescence signal from the exogenousfluorescence agent over the course of injection and renal elimination istypically expected to be about 10% of the magnitude of the signalreceived during illumination at the excitation wavelength by the firstlight source 218, assuming that the exogenous fluorescence agent isMB-102 introduced into the patient 202 at a dose level of about 4μmol/kg of patient weight. In an aspect, if the expected detector signalreceived during illumination at maximum LED intensity and with thedetector gain set to the high setting remains below 10% of the range ofthe detector ADC, the detector gain for that measurement be increased byten-fold. In another aspect, the saturation condition may persist for apre-defined period of time including, but not limited to, a 30-secondperiod before adjustments are made to the detector gain or LED power toavoid reacting to spurious signal spikes.

In another aspect, the light detector control unit 232 may adjust thedetector gain to a lower gain level if the detected light signals fromone of the light detectors 222/224 exceed a threshold percentage of themaximum ADC range to avoid signal saturation. Although the highestthreshold percentage of the maximum ADC range associated with signalsaturation is 100%, the onset of severe detector non-linearity takesplace at threshold percentages of about 40% or more, and mild detectornon-linearity occurs at threshold percentages in excess of about 15%. Invarious aspects, the threshold percentage of the maximum ADC range maybe 40%, 35%, 30%, 25%, 20%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, or 5% of the maximum ADC range. In one aspect, if thedetected light signals from one of the light detectors 222/224 exceedabout 8% of the maximum ADC range, the gain setting will be adjusted. Byway of non-limiting example, if the detector gain on the nearlysaturated signal is high, it will be adjusted to low. If the currentdetector gain is set to low and the corresponding detected light signalremains above the threshold percentage of the maximum ADC range, the LEDoutput power setting of the corresponding LED light source may bereduced ten-fold.

In an aspect, the light detector control unit 232 may receive one ormore feedback measurements used to modulate the plurality of detectorsignals to compensate for variations in the performance of the lightdetectors due to variations in temperature and/or light source output.Non-limiting examples of feedback measurements used by the lightdetector control unit 232 include: light output of the light sources218/220 measured within the source well 902 by the first monitorphotodiode 904 and the second monitor photodiode 906, respectively (seeFIG. 11), temperatures of the light detectors 222/224 measured by afirst temperature sensor 1106, LED temperatures measured by a secondtemperature sensor 1108, temperature of the sensor head housing measuredby a third temperature sensor 1128, LED supply current from the LEDcurrent source 1126, and any other feedback measurement relevant tomonitoring the performance of light detectors 222/224.

In various aspects, the light detectors 222/224 may be silicon photonmultiplier (SPM) detectors that may include low-noise internalamplification, and may function at lower light levels relative to otherlight sensor devices such as PIN photodiodes. The detector signalgenerated by the SPM detectors 222/224 may be amplified usingtransimpedance amplifiers 1120/1118, respectively (see FIG. 11) totranslate a current generated by each SPM light detector 222/224 into ameasurable detector voltage. The transimpedance amplifier 1118 on thesecond SPM light detector 224 (i.e. detects filtered lights at theexcitation wavelength only) may include a switchable detector gain thatmay select a low gain configured to detect a larger dynamic range forfluorescence measurements when the first LED light source 218 isactivated to produce light at the emission wavelength. The switchabledetector gain that may further select a high gain setting for the secondSPM light detector 224 when the second light source 220 is inactive toenhance the sensitivity of the second SPM light detector 224 during thephase of the detection cycle when light at the emission wavelengthproduced by the exogenous fluorescent agent within the tissues of thepatient 202 is detected, to ensure that the expected dark current fromthe second SPM light detector 224 occupies less than ¼ of the total ADCoutput range. In one aspect, the second transimpedance amplifier of thesecond SPM light detector 224 may include a low detector gain configuredto provide a transimpedance gain of about 4 kΩ corresponding to abouttwice the value of the transimpedance resistor due to differentialoperation, and may further include a high detector gain configured toprovide a transimpedance gain of about 40 kΩ. In another aspect, thefirst transimpedance amplifier of the first SPM light detector 222 mayinclude a fixed detector gain configured to provide a transimpedancegain of about 2 kΩ.

iii) Acquisition Unit

Referring again to FIG. 2, the controller 212 may further include anacquisition unit 234 in various aspects. The acquisition unit 234 may beconfigured to receive a plurality of signals from the light sources218/220, light detectors 222/224, and additional light detectors 226 andadditional temperature sensors 228 and processing the plurality ofsignals to produce one or more raw signals including, but not limitedto, raw fluorescence signals encoding the intensity of fluorescencedetected by the second light detector 224 during illumination at theexcitation wavelength, and raw internal reflectance signalscorresponding to the intensity of light at the excitation wavelengthdetected by the first light detector 222 during illumination at theexcitation wavelength as well as the intensity of light at the emissionwavelength detected by the both light detectors 222/224 duringillumination at the emission wavelength.

The plurality of signals received from the various sensors and devicesdescribed herein above are typically analog signals including, but notlimited to, electrical voltages and currents. In various aspects, theacquisition unit 234 may enable the transmission of the analog signalsto one or more analog-to-digital converters (ADCs) to convert the analogsignals into digital signals for subsequent processing by the processingunit 236. FIG. 11 is a schematic diagram of a circuit 1100 illustratingthe arrangement of various electrical devices and components of thesensor head 204. In one aspect, the analog signals encoding theintensity of light detected by the first light detector 222 and thesecond light detector 224 may be received by a first ADC 1102.

In various aspects, the analog signals produced by the light detectors222/224 and various monitor sensors may be digitized using at least one24-bit Sigma-Delta ADC. Referring again to FIG. 11, analog signalsencoding the measurements from time-sensitive sensors may be digitizedusing a high-speed 24-bit Sigma-Delta ADC 1102 in one aspect. In thisaspect, time-sensitive sensors include sensors associated with theproduction and detection of light pulses characterized by potentiallyrapidly-changing signals. Non-limiting examples of time-sensitivesensors of the system 200 include: first and second light detectors1118/1120, and first and second monitor photodiodes 904/906. In anotheraspect, analog signals encoding the measurements from lesstime-sensitive sensors may be digitized using a low-speed 24-bitSigma-Delta ADC 1104. In this other aspect, the less time-sensitivesensors include sensors associated with monitoring system conditionscharacterized by typically slow-changing signals including, but notlimited to, temperatures of various system components and/or regions.Non-limiting examples of less time-sensitive sensors of the system 200include: a first and second thermistor 1106/1108 configured to monitorthe temperatures of the light sensors 222/224 and light sources 218/220,respectively, and a third temperature sensor 1128 configured to monitora temperature of the housing 600 of the sensor head 204.

In various aspects, the acquisition unit 234 may be further configuredto enable synchronous detection of light by detectors 222/224. Withoutbeing limited to any particular theory, synchronous detection methodsare thought to reject noise from the detector signals associated withthe detection of light produced by the light sources 118/120 andfluorescence produced by the exogenous fluorescent agents within thetissues of the patient 202 by distinguishing the detector signals fromnoise associated with the detection of ambient light or other sources ofinterference.

FIG. 12 is a schematic illustration of a synchronous detection method inone aspect. Referring to FIG. 11 and FIG. 12, the waveform generator/FPA1122 may generate a digital square wave 1202 that is received by the DAC1124, and the resulting analog-converted square wave is received by theLED current source 1126. The resulting current produced by the LEDcurrent source 1126, also characterized by a waveform proportional tothe analog-converted square wave drives LED light sources 218/220. Thelight produced by LED light sources 218/220, after passing through thetissues of the patient 202 are detected, along with the fluorescenceproduced by the endogenous fluorescent agent, by the light detectors222/224 and are digitized by the high-speed ADC 1102.

Referring again to FIG. 11 and FIG. 12, the digital square wave 1202generated by the waveform generator/FPA 1122 may also be converted by aDAC 1110 (see FIG. 11) to an in-phase reference sine wave 1210 and anout-of-phase/quadrature reference cosine wave 1212. In an aspect, thedigitized detector signals from the ADC 1102 and the in-phase referencesine wave 1210 may be sampled and subjected to signed multiplication ata first multiplier 1214 to generate a plurality of in-phase modulatedsignals. In addition, the digitized detector signals and the quadraturereference cosine wave 1212 may be sampled and subjected to signedmultiplication at a second multiplier 1216 to generate a plurality ofquadrature (out-of-phase) modulated signals. In this aspect, theacquisition unit 234 may delay the samples from the reference waves1210/1214 by an amount equivalent to the relative delay between the DAC1124 generating the reference waves 1210/1214 and the ADC 1102digitizing the detector signals to synchronize the reference waves1210/1214 to the detector data being acquired.

Referring again to FIG. 12, the in-phase modulated signals may be summedin a first accumulator 1218 to generate an in-phase intensity signal1224. Similarly, the quadrature modulated signals may be summed in athird accumulator 1222 to generate a quadrature intensity signal 1228.The raw digitized detector signal may also be summed in a secondaccumulator 1220 to generate an average intensity signal 1226. Inaddition, the in-phase intensity signal 1224 and the quadratureintensity signal 1228 may be root-sum squared to generate a magnitudesignal 1230.

Without being limited to any particular theory, the integration intervalof the accumulators 1218/1220/1222 may correspond to an integer numberof modulation cycles (corresponding to cycles of the digital square wave1202) to avoid a bias on the measured signal. The phase accumulators1218/1220/1222 used to control the synchronous detection operates oninteger numbers, but the sample clock frequency and the modulationfrequency are not integer-divisible, so the number of cycles is notexactly an integer. However, the error associated with this mismatch maybe minimized by adjusting the actual modulation frequency to match asclosely as possible with the achievable sampling intervals andallocating an appropriate number of bits to the phase accumulator. Inone aspect, the error associated with the mismatch between themodulation frequency and the sampling intervals may be on the order ofabout one part in 10⁶.

In one aspect, the digital square wave 1202 used to modulate the LEDlight sources 218/220 and to enable synchronous detection method asdescribed herein above is produced at a frequency of about 1 kHz.Without being limited to any particular theory, a square wave wasselected as the modulating waveform to enable an enhancement in signalto noise ratio (SNR), as compared to a pure sinusoidal wave as themodulating waveform for the same peak power level.

In another aspect, the acquisition unit 234 may be further configured toenable demodulation of the in-phase intensity signal 1224, averageintensity signal 1226, and quadrature intensity signal 1228. In oneaspect, the acquisition unit 234 may pick out each component at thefundamental harmonic, which is characterized by an amplitude that is(4/π) times larger than the amplitude of the square wave 1202 used tomodulate the intensity signals 1224/1226/1228. In various aspects, toreject 50/60 Hz electrical noise generated by the alternating currentelectrical power sources, and corresponding 100/120 Hz optical noisegenerated by ambient light sources powered from those electrical powersources, the integration period of the accumulators 1218/1220/1222 maybe selected to be a multiple of 100 ms. In these various aspects, thisselected integration period ensures that integration by the accumulators1218/1220/1222 occurs over an integer number of cycles for the 50, 60,100, and 120 Hz signals.

iv) Processing Unit

Referring again to FIG. 2, the controller 212 may further include aprocessing unit 236 configured to apply corrections to the demodulateddetector signals and to transform a selected portion of the correcteddetector signals into a measure of renal function in various aspects.FIG. 13 is a block diagram illustrating the subunits of processing unit236 in an aspect. Referring to FIG. 13, the processing unit 236 mayinclude a pre-processing subunit 1302 configured to determine andcorrect the detector signals to remove signal artifacts associated witha variety of confounding effects including, but not limited to,physiologically-induced signal variations, variations in power suppliedto the light sources 218/220, non-linearities in detector response,ambient temperature variation, and tissue heterogeneity. The processingunit 236 may further include a baseline subtraction subunit 1304configured to remove the portion of the detector signals attributable toextraneous factors such as autofluorescence of the tissues and/orleakage of light at the excitation wavelength through the optical filter244 of the second light detector 224. The processing unit 236 mayadditionally include a diffuse reflectance correction subunit 1306configured to enable a method of applying a diffuse reflectancecorrection method to remove the effects of the diffuse reflectance oflight within the tissues of the patient 202. The processing unit 236 mayfurther include a post-equilibrium selection subunit 1308 configured toselect a portion of the detector data associated with the post-agentadministration period for subsequent analysis to determine renalfunction of the patient. The processing unit 236 may further include anRDTC calculation subunit 1310 configured to transform the detectorsignals obtained over the post-agent administration period to produce arenal decay time constant indicative of the renal function of thepatient. The processing unit 236 may also include a fault detectionsubunit 1312 configured to monitor the magnitudes of the detectorsignals to detect any malfunctions of the system.

a) Pre-Processing Subunit

In one aspect, the raw signals corresponding to the light intensitydetected by light detectors 222/224 corresponding to illumination by thefirst light source 218 and the second light source 220 at the excitationand emission wavelength, respectively, are pre-processed using variousmodules of the pre-processing subunit 1302 to remove the effects of aplurality of confounding factors from the raw signals, resulting insignals that more accurately reflect the underlying specific signals ofinterest.

By way of several non-limiting examples, the intensity of light producedby a light source may vary due to one or more of a plurality of factorsincluding, but not limited to: fluctuations in the electrical currentsupplied to the light source and variations in the ambient temperatureof the light source. Light characterized by two or more wavelengthsemanating from the same source aperture of the sensor head may not sharethe same path to the same detector. The detectors may havethermally-dependent sensitivity and gain. Further, the optical filterassociated with the second light detector 224 may havetemperature-dependent transmission properties.

In one aspect, the pre-processing subunit 1302 is configured to processthe raw signals corresponding to light intensities detected by the firstand second light detectors 222/224 in order to remove one or more of themeasurement errors associated with the devices and elements of thesystem 200 and patient-specific factors including, but not limited to,the plurality of factors described above. FIG. 22A is a block diagramillustrating the modules of the pre-processing subunit 1302 in oneaspect. FIG. 22B is a block diagram illustrating the modules of thepre-processing subunit 1302 a in a second aspect.

In one aspect, illustrated in FIG. 22A, the pre-processing subunit1302 1) resamples the signals using the methods of the resampling module2202 as described below, 2) removes saturated detector signals using themethods of the detector output saturation detection and removal module2204 as described below, 3) corrects for temperature-dependent detectorgain using the methods of the detector temperature correction module2206 described below, 4) corrects the signals for instrument lightdirectionality using the methods of the light directionality correctionmodule 2208 described below, 5) corrects the signals for filterthroughput and temperature-dependent variation of fluorescence lightusing the methods of the filter throughput temperature correction(emission) module 2212 described below, 6) corrects for tissueheterogeneity using the methods of the tissue heterogeneity correctionmodule 2216 described below, 7) corrects the signals for filterthroughput and temperature-dependent variation of excitation light andsignal decomposition using the methods of the filter throughputtemperature correction (excitation) module and signal decompositionmodule 2214 as described below, 8) corrects for optical power variationusing the methods of the fractional photon normalization module 2218 asdescribed below.

In one aspect, illustrated in FIG. 22B, the pre-processing subunit 1302a calculates signal magnitudes using the methods of the detectortemperature correction module 2206 a as described below, resamples thesignals using the methods of the resampling module 2202 a as describedbelow, removes saturated samples using the methods of the detectoroutput saturation detection and removal module 2204 a as describedbelow, corrects the signals for temperature-dependent detector gainusing the methods of the detector temperature correction module 2206 adescribed below, corrects the signals for optical power variation usingthe methods of the fractional photon normalization module 2218 a asdescribed below, corrects for excitation light leakthrough onto themeasured fluorescence signal using the filter throughput temperaturecorrection (excitation) module and signal decomposition module 2214 a asdescribed below, and corrects for fluorescence light leakthrough ontothe measured excitation diffuse reflectance signal using the filterthroughput temperature correction (emission) module 2212 a as describedbelow.

—Resampling Module

Referring to FIG. 22A and FIG. 22B the pre-processing subunit 1302/1302a in various aspects includes a resampling module 2202/2202 a configuredto reduce signal variations associated with physiological processes ofthe patient 202 including, but not limited to, heartbeat and breathing.Typically, an acquisition sequence is characterized by alternatinginterval of illumination at the excitation and emission separated byintervals of no illumination (i.e. dark intervals). Although bothillumination intervals (excitation/emission) are time-stamped with thesame time-stamp value as described above, the dark interval between theexcitation and emission illumination intervals results in a separationinterval between the excitation and emission illumination intervals.Without being limited to any particular theory, if the separationinterval associated with an acquisition sequence is on the order of aseparation interval between physiological events, such as heartbeats orrespiration, physiological noise may be introduced to the signals. Invarious aspects, this physiological noise may be reduced by resamplingthe signals associated with the excitation and emission illumination tooverlap prior to subsequent processing of the signals.

By way of non-limiting example, a sample sequence may include a 100 msdark interval, a 100 ms interval of illumination at the excitatorywavelength, a second 100 ms dark interval, and a 100 ms interval ofillumination at the emission wavelength. Each sample packet is loggedwith a single timestamp, and each sample packet is separated by a 400 msinterval. Because physiological signal variations, such as fromheartbeats, occur on this same timescale, the 200 ms difference betweensignal acquisition associated with the excitatory and emissionwavelengths becomes apparent in the signals. This physiological signalnoise may be reduced using the pre-processing subunit 1302 by firstresampling the signals associated with illumination at the excitatoryand emission wavelength illumination to overlap prior to performing anyadditional signal processing as described below. In this non-limitingexample, the signals associated with illumination at the excitatorywavelength may be shifted forward by 100 ms and the signals associatedwith illumination at the emission wavelength may be shifted backwards by100 ms, resulting in an overlap of the signals.

In various aspects, the resampling module 2202 performs resampling asdescribed above on signals detected by both the first and seconddetectors 222/224. In one aspect, the resampling module 2202 functionsas a form of low-pass filter.

—Detector Output Saturation Detection and Removal Module

Referring again to FIG. 22A and FIG. 22B the pre-processing subunit1302/1302 a in various aspects includes a detector output saturationdetection and removal module 2204/2204 a configured to detect and removesignal values that fall outside the detection range of the lightdetectors 222/224. In one aspect, the pre-processing subunit 1302compares the detected signals to the maximum ADC signal. If any signalfalls within a threshold range of the maximum ADC signal using theaverage or peak signal value, the detector output saturation detectionand removal module 2204 identifies and removes that value from furtherprocessing.

—Detector Temperature Correction Module

FIG. 22A and FIG. 22B the pre-processing subunit 1302/1302 a in variousaspects includes a detector temperature correction module 2206/2206 aconfigured to enable a temperature correction to compensate for thethermal sensitivity of the light detectors 222/224. In one aspect, theintrinsic detector gain for a silicon photomultiplier (SPM) devicetypically used as a light detector is proportional to the differencebetween the device breakdown voltage and the bias voltage applied by thebias voltage generator 1112 (see FIG. 11), referred to herein as anovervoltage. In this aspect, the breakdown voltage varies withtemperature in a well-characterized manner. In one aspect, thetemperature correction accounts for both this internal detector gainvariation and additionally temperature-related variation in the photondetection efficiency.

In one aspect, the temperature correction may be a scaling correctionapplied to the detector measurements in which the scaling correction isbased on a measured detector temperature. In an aspect, the measuredlight detector signals may be divided by the calculated gain G(t) toremove the temperature dependency. The scaling correction G(t) may becalculated according to Eqn. (2):G(T)=C _(v) ·V _(bias) −V _(breakdown)(1+C _(T))^(T-T) ⁰   Eqn. (2)

In Eqn. (2), the monitor temperature T is obtained from a firsttemperature sensor 1106 (see FIG. 11) configured to monitor thetemperature of the sensors 222/224. The bias voltage (V_(bias)) may bemeasured by the bias voltage generator 1112. The breakdown voltage(V_(breakdown)) and reference temperature (T₀) are constants specific tothe particular light detector device included in the system 200. By wayof non-limiting example, if the light detectors 222/224 are siliconphotomultiplier (SPM) devices, V_(breakdown) may be 24.5 V and T₀ may be21 degrees C. In another aspect, the coefficients C_(v) and C_(T) usedin Eqn. (2) may be derived empirically based on measurements obtainedusing a constant phantom over an ambient temperature ranging from about18 degrees C. to about 26 degrees C.

In another aspect, the temperature portion of the gain correction isdetermined by the Eqns. (3)-(5).

$\begin{matrix}{G_{useCase} = {{C_{v} \cdot V_{{bias}_{measured}}} - {V_{breakdown}\left( {1 + C_{T}} \right)}^{T_{measured} - T_{0}}}} & {{Eqn}.\mspace{14mu}(3)} \\{G_{nominal} = {{C_{v} \cdot V_{{bias}_{nominal}}} - {V_{breakdown}\left( {1 + C_{T}} \right)}^{T_{nominal} - T_{0}}}} & {{Eqn}.\mspace{14mu}(4)} \\{G_{correction} = \frac{G_{useCase}}{G_{nominal}}} & {{Eqn}.\mspace{14mu}(5)}\end{matrix}$

This gain correction can be applied to each of the signal magnitudes asmeasured by the first and second light detectors 222/224 as follows:

$\begin{matrix}{{SPMmagnitude}_{corrected} = \frac{SPMmagnitude}{G_{correction}}} & {{Eqn}.\mspace{14mu}(6)}\end{matrix}$

In an aspect, the magnitudes of the temperature-corrected measurementsfrom each detector and monitor photodiode are calculated from the rootsum-squares of the in-phase intensity signals 1224 (I) and quadratureintensity signals 1228 (Q) according to Eqn. (1):M=√{square root over (I ² +Q ²)}  Eqn. (1)

The signal magnitudes from the light detectors 222/224 calculated usingEqn. (1) are normalized by the monitor photodiode magnitude for eachmeasurement set corresponding to the measurements obtained duringillumination by one of the LED light sources 218/220 at either theexcitation or emission wavelength. In one aspect, if one photodiode ispositioned in the source well 902, the single photodiode magnitude fromthe corresponding measurement set is used for this normalization. Inanother aspect, if two monitor photodiodes 904/906 are positioned in thesame source well 902 as both LED light sources 218/220 (see FIG. 9), theaverage of the two monitor photodiode magnitudes from the correspondingmeasurement set is used for this normalization.

In an aspect, the in-phase intensity signal 1224, quadrature intensitysignal 1228, and average intensity signal 1226 (see FIG. 12) are furtherprocessed for the number of accumulated samples and ADC scaling suchthat the intensity signals 1224/1226/1228 are returned as fraction ofthe full range of the high-speed ADC 1102 (i.e. ranging from a minimumof 0 to a maximum of 1). The measurements of the monitor photodiodes904/906 (see FIG. 11) are similarly scaled as a fraction of the fullrange of the low-speed ADC 1104.

In one aspect, G_(correction) may incorporate a power correction tocorrect for the effects of fluctuations in the LED power supply. In thisaspect, the signals from the first monitor photodiode 904 and the secondmonitor photodiode 906 are calibrated by measuring optical output powerwith a power meter as light intensities from the light sources 218/220are varied. The calibration coefficients for each light source 218/220,C_(source1) and C_(source2), are calculated as detector-measuredmilliWatts per recorded monitor photodiode signal value. C_(source) andC_(source2) are used to determine the absolute light output into tissueat each wavelength.

Referring again to FIG. 22B, the detector temperature correction module2206 a corrects signal magnitudes for the varying intensity of the LEDsby normalizing the temperature-corrected detected signals using the LEDoutput signal PD_(magnitude) measured by the first monitor photodiode904 and/or the second monitor photodiode 906. In this case, theG_(correction) variable for each light source 218/220 from above isamended as follows:

$\begin{matrix}{G_{correction} = {\frac{G_{useCase}}{G_{nominal}}*{PD}_{magnitude}}} & {{Eqn}.\mspace{14mu}(7)}\end{matrix}$—Light Directionality Correction Module

Referring again to FIG. 22A, the pre-processing subunit 1302 in thisaspect includes a light directionality correction module 2208 configuredto enable a correction to variations in the detected signals associatedwith differences in the scattering and absorption of light of differentwavelengths through the tissues of the patient 202 during dataacquisition. In one aspect, a correction term for light directionalitymay be measured by acquiring data from one or more homogeneous tissuephantoms and using a sensor configuration in which no emission filtersare present. The ratio of the signals detected by the first lightdetector 222 (Det1) and the signals detected by the second lightdetector 224 (Det2) measured are used to determine a coefficient G_(ex)or G_(em) for signals obtained in association with illumination by lightat the excitation and emission wavelengths, respectively. Thecoefficients are used to modify the signal detected by the first lightdetector 222. In one aspect, the correction of the signals acquired in ahomogeneous medium by the first light detector 222 using thecoefficients G_(ex) or G_(em) render the signals measured by the firstand second detectors 222/224, as equivalent to within 20% of oneanother. In other aspects, the correction of the signals acquired in ahomogeneous medium by the first light detector 222 using thecoefficients G_(ex) or G_(em) render the signals measured by the firstand second detectors 222/224 as equivalent to within about 10%, towithin about 5%, to within about 2%, and to within about 1%.

—Detector Non-Linear Response Correction Module

Referring again to FIG. 22A, the pre-processing subunit 1302 in thisaspect includes a detector non-linear response correction module 2210configured to enable a correction to variations in the detected signalsassociated with non-linear response of the detectors. In this aspect, acalibration curve based on average data may be used to scale themagnitude data obtained by the detectors 222/224.

—Filter Throughput Temperature Correction (Emission) Module

Referring again to FIG. 22A, the pre-processing subunit 1302 in thisaspect includes a filter throughput temperature correction (emission)module 2212 configured to enable a correction to variations in thedetected signals associated with temperature-dependent opticalproperties of the optical filter 244 associated with the second lightdetector 224 during emission-wavelength illumination. In this aspect,the signals Det2 detected by the second light detector 224 may becorrected according to Eqn. (8):

$\begin{matrix}{{{Det}\; 2} = \frac{{{Det}\; 2} - {{Det}\; 2\left( {C_{{emF},{slopeT}}\left( {T - T_{nom}} \right)} \right)}}{C_{{emF},{nom}}}} & {{Eqn}.\mspace{14mu}(8)}\end{matrix}$

In various aspects, the signal Det2 measured by the second lightdetector 224 may be monitored while ambient temperature is cycled over arange including the operating temperature range or a large enough subsetof the range to adequately determine the temperature-dependence of theemission filter. These data are acquired with the optical filter 244installed on the second light detector 224 from a homogeneous,non-fluorescent phantom. Further, simultaneous measurements aremonitored from the first light detector 222, and a ratio of themeasurements Det2/Det1 is determined. The nominal filter coefficient,C_(emF,nom) is calculated as the nominal ratio of Det2/Det1 obtained ata nominal operating temperature T_(nom). In this aspect, the coefficientC_(emF,slopeT) is obtained from the slope of Det2/Det1 obtained over arange of ambient temperatures during emission-wavelength illumination ofthe homogeneous, non-fluorescent phantom.

—Tissue Heterogeneity Correction Module

Referring again to FIG. 22A, the pre-processing subunit 1302 in thisaspect includes a tissue heterogeneity correction module 2216 configuredto enable a correction to variations in the detected signals associatedwith heterogeneity of the tissues intervening between the first region206 illuminated by light sources 218/220 and the second and thirdregions 208/210 at which the light detectors 222/224 are positioned. Inthis aspect, the signal Det1 corrected for light directionality by thelight directionality correction module 2208 and the signal Det2corrected for filter effects by the filter throughput temperaturecorrection (emission) module 2212 are used to calculate C_(hetero), acoefficient to correct for tissue heterogeneity, according to Eqn. (9):C _(hetero) =Det2/Det1  Eqn. (9)—Filter Throughput Temperature Correction (Excitation) and SignalDecomposition Module

Referring again to FIG. 22A, the pre-processing subunit 1302 in thisaspect includes a filter throughput temperature correction (excitation)module and signal decomposition module 2214 configured to enable acorrection to variations in the detected signals associated withtemperature-dependent optical properties of the optical filter 244associated with the second light detector 224 duringexcitation-wavelength illumination. In this aspect, because the emissionfilter is configured to block light at the excitation wavelength, thefilter throughput temperature correction (excitation) module and signaldecomposition module 2214 performs a correction to variance to theamount of excitation light leakthrough due to temperature-relatedchanges in the optical properties of the optical filter 244. Further,the filter throughput temperature correction (excitation) module andsignal decomposition module 2214 enables corrections of the signalsmeasured by the first light detector 222 during excitation-wavelengthillumination due to the presence of fluorescence induced by theexcitation-wavelength illumination superimposed over the portion of thesignal associated with the excitation-wavelength illumination.

In this aspect, the effects of temperature-dependent variation onleakthrough of excitation-wavelength by the optical filter 244 arecalculated as expressed in Eqn. (10):C _(exLT) =C _(exLT,nom) +C _(exLT,slopeT)(T−T _(nom))  Eqn. (10)

In this aspect, C_(exLT,nom) is calculated from the ratio of signalsDet1 and Det2 measured from a homogeneous, non-fluorescent phantom atthe nominal operating temperature T_(nom) during excitation-wavelengthillumination. C_(exLT,slopeT) is calculated as the slope of the signalDet2 measured from a homogeneous, non-fluorescent phantom at a range ofoperating temperatures T during excitation-wavelength illumination.

In this aspect, the filter throughput temperature correction(excitation) module and signal decomposition module 2214 furtherperforms a signal extraction to isolate portions of the detected signalsassociated with diffuse reflectance of the excitation-wavelengthillumination and fluorescence. DR_(ex2), which is the amount ofexcitation light impingent on the second light detector 224 in theabsence of an optical filter 244, is not measurable, due to the presenceof the optical filter 244. Further, the signal Det1 measured by thefirst light detector 222 is a composite signal from both diffusereflectance of the excitation-wavelength illumination DR_(ex1) andfluorescence Flr1. C_(Hetero) is obtained using the tissue heterogeneitycorrection module 2216 as described above. The underlying signals areextracted by use of the following system of equations:Det ₂ =C _(exLT) DR _(ex2) +Flr2  Eqn. (11)Det ₁ =DR _(ex1) +Flr ₁  Eqn. (12)Flr ₂ =C _(Hetero) Flr ₁  Eqn. (13)DR _(ex2) =C _(Hetero) DR _(ex1)  Eqn. (14)

In this aspect, Flr₂ is determined by solving the above system ofequations using only measurable signals Det1 and Det2 as demonstratedbelow:

$\begin{matrix}{{Det}_{2} = {{C_{exLT}C_{Hetero}{DR}_{{ex}\; 1}} + {Flr}_{2}}} & {{Eqn}.\mspace{14mu}(15)} \\{{Det}_{2} = {{C_{exLT}{C_{Hetero}\left( {{Det}_{1} - {Flr}_{1}} \right)}} + {Flr}_{2}}} & {{Eqn}.\mspace{14mu}(16)} \\{{Det}_{2} = {{C_{exLT}C_{Hetero}{Det}_{1}} - {C_{exLT}C_{Hetero}{Flr}_{1}} + {Flr}_{2}}} & {{Eqn}.\mspace{14mu}(17)} \\{{{Det}_{2} - {C_{exLT}C_{Hetero}{Det}_{1}}} = {{Flr}_{2}\left( {1 - C_{exLT}} \right)}} & {{Eqn}.\mspace{14mu}(18)} \\{{Flr}_{2} = \frac{{Det}_{2} - {C_{exLT}C_{Hetero}{Det}_{1}}}{1 - C_{exLT}}} & {{Eqn}.\mspace{14mu}(19)}\end{matrix}$

In this aspect, once Flr2 is obtained as described above, the othersignals Flr₁, DR_(ex1), and DR_(ex2) may be readily obtained throughinsertion into the system of equations (Eqns. (11)-(14)) presentedabove.

—Fractional Photon Normalization Module

Referring again to FIG. 22A, the pre-processing subunit 1302 in thisaspect includes a fractional photon normalization module 2218 configuredto convert the detector signals, after preprocessing as described above,into units of fractional photons for use in subsequent backgroundsubtraction and intrinsic fluorescence correction algorithms asdescribed herein. In this aspect, the detector signals may be convertedto photocurrent by reversing the scaling associated with the ADC and thetransimpedance amplifier used to acquire the detected signals to obtainthe signals in units of photocurrents. Once photocurrent is obtained, adetector responsivity supplied by the light detector's manufacturer isused to convert the detector photocurrents to units of Watts. Thedetector signals in Watts are then ratioed to the source power in Wattsas measured by additional light detectors 226 used to monitor the outputof the light sources 218/220 to obtain the number of fractional photonsdetected.

—Optical Power Correction Module

Referring again to FIG. 22A and FIG. 22B, the pre-processing subunit1302/1302 a in this aspect includes a fractional photon normalizationmodule 2218/2218 a configured to convert the detector signals, afterpreprocessing as described above, into units of fractional photons foruse in subsequent background subtraction and intrinsic fluorescencecorrection algorithms as described herein. In this aspect, the detectorsignals may be converted to photocurrent by reversing the scalingassociated with the ADC and the transimpedance amplifier used to acquirethe detected signals to obtain the signals in units of photocurrents.Once photocurrent is obtained, a detector responsivity supplied by thelight detector's manufacturer is used to convert the detectorphotocurrents to units of Watts. The detector signals in Watts are thenratioed to the source power in Watts as measured by additional lightdetectors 226 used to monitor the output of the light sources 218/220 toobtain the number of fractional photons detected.

—Excitation Light Leakthrough Subtraction Module

Referring again to FIG. 22B, the pre-processing subunit 1302 a in thisaspect includes a fractional photon normalization module 2222 configuredto perform an excitation leakthrough subtraction on the Flr_(meas)signal. To arrive at a fluorescence signal due only to fluorescentphotons (Flr_(photons)), an excitation leakthrough subtraction isperformed. To remove the contribution of excitation light, theexcitation leakthrough is taken to be a fraction of the diffusereflectance excitation (DR_(ex) _(meas) ) signal, where a universalcalibration factor, C_(ExLT), determines the fraction of the signal tosubtract from Flr_(meas) as expressed below:ExLT=C _(ExLT) *DR _(ex) _(meas)where C_(ExLT) is a calibration factor that is obtained by computing theratio between the excitation light detected by both detectors on anon-fluorescing optical phantom as described below:

$C_{ExLT} = \frac{{Flr}_{meas}}{{DR}_{{ex}_{meas}}}$

This signal is then subtracted from Flr_(meas) to provide a fluorescencesignal due only to fluorescent photons as expressed below:Flr _(photons) =Flr _(meas) −ExLT—Fluorescence Light Leakthrough Subtraction Module

Referring again to FIG. 22B, the pre-processing subunit 1302 a in thisaspect includes a fluorescence light leakthrough subtraction module 2224a configured to perform a fluorescence leakthrough subtraction on theFlr_(meas) signal. To obtain the diffuse reflectance, defined herein asthe excitation signal due to only excitation photons (DRex_(photons)), afluorescence leakthrough subtraction is performed. To remove thefluorescence leakthrough, a calibration factor, C_(FlrLT), wasdetermined based on the relationship between the amount of fluorescenceleakthrough observed on a database of human subject data and tissueheterogeneity as measured by the relationship between the diffusereflectance, emission signals

$\left( \frac{DRemFilt}{DRem} \right).$The relationship is a linear relation as expressed below:

$C_{FlrLT} = {{p\; 1*\left( \frac{DREM}{DRemFilt} \right)} + {p\; 2}}$where p1 and p2 are approximately 0.61 and 0.01, respectively, in oneaspect, as determined by the above-mentioned relationship. In anotheraspect, p1 and p2 may assume any other value without limitation asdefined by the above relationship.

The DRex_(photons) signal is then calculated by subtracting thisfraction of measured fluorescence from the diffuse reflectanceexcitation signal, as follows:DRex _(photons) =DR _(ex) _(meas) −Flr _(meas) *C _(FLrLT)b) Baseline Subtraction Subunit

Referring again to FIG. 13, the processing unit 236 further includes abaseline subtraction subunit 1304. In an aspect, the baselinesubtraction subunit 1304 subtracts a baseline signal from the lightdetector measurements to correct for the effects of autofluorescence andlight leakage. The baseline period, as used herein, refers to an initialtime period of measurements obtained prior to injection of the exogenousfluorescent agent. During the baseline period, the fluorescence signalmeasured by the system 200 may be assumed to associated with tissueautofluorescence and/or excitation light from the LED light sources218/220 leaking through the absorption filter 244 of the second lightdetector 224. In an aspect, the average signal measured during thebaseline period, referred to herein as a baseline signal, may besubtracted from subsequent fluorescence measurements to yield ameasurement associated solely with the fluorescence produced by theexogenous fluorescent agent within the tissues of the patient.

In another aspect, the corrections for excitation light leak-through andautofluorescence may be implemented separately. In this other aspect, asubtraction of the effects of excitation light leak-through may beperformed prior to the diffuse reflectance correction described hereinbelow, and a subtraction of the effects of autofluorescence may beperformed after the diffuse reflectance correction.

c) Diffuse Reflectance Correction Subunit

Referring again to FIG. 13, the processing unit 236 further includes adiffuse reflectance correction subunit 1306. In an aspect, the diffusereflectance correction subunit 1306 may correct the measuredfluorescence data to remove the effects of changes to the opticalproperties (absorption and scattering) of the tissues of the patient 202during monitoring of renal extraction of an exogenous fluorescent agentwithin the tissues of a patient. As described herein above, the opticalproperties of the tissues may change due to any one or more factorsincluding, but not limited to: vasodilation, vasoconstriction, oxygensaturation, hydration, edema, and any other suitable factor within theregion of interest monitored by the system, associated with changes inthe concentrations of endogenous chromophores such as hemoglobin andmelanin.

Without being limited to any particular theory, the fluorescencemeasurements obtained by the system 200 that are used to determine renalfunction include emission-wavelength photons that are detected by thesecond (filtered) light detector 224. These emission-wavelength photonsare emitted by the exogenous fluorescence agent introduced into thetissues of the patient in response to illumination byexcitation-wavelength photons. The emission-wavelength photons travelfrom the fluorescence source (i.e. the exogenous fluorescence agent) tothe second (filtered) light detector 224 through third region 210 of thepatient's skin. However, the emission-wavelength light that is detectedby the second (filtered) light detector 224 may also includeautofluorescence emitted by endogenous fluorophores such as keratin andcollagen within the tissues of the patient, as well as leak-through ofexcitatory-wavelength light through the optical filter 244 of the secondlight detector 224. The excitation-wavelength photons that inducefluorescence of the exogenous fluorescent agent are produced by thefirst light source 218 and are directed into the first region 206 of thepatient's skin. If the optical properties of the patient's skin(scattering and/or absorption) varies over the time interval at whichthe detector data used to determine renal function is acquired (i.e.from a few hours to about 24 hours or more), the accuracy of thefluorescence measurements may be impacted, as discussed previouslyabove.

During each measurement cycle in an aspect, the system 200 may directlight into the first region 206 of the patient's skin with a pulse ofemission-wavelength light and a pulse of excitation-wavelength light inan alternating series and may detect all light emerging from the secondregion of the patients skin using the first (unfiltered) light detector222 and a portion of the light emerging from the third region 210 of thepatient's skin using the second (filtered) light detector 224. The lightintensity detected by each combination of excitation and emissionwavelength illumination of the first region 206 and detection by theunfiltered/filtered light detectors 222/224 contain information not onlyabout the concentration of the exogenous fluorescent agent in thepatient's tissues, but also information about the optical properties ofthe patient's skin.

TABLE 2 Light Detector Measurements After Temperature and PowerFluctuation Corrections First (Reference) Second (Primary) IlluminationLight Detector Light Detector wavelength Unfiltered FilteredExcitation-wavelength Flr_(meas) Flr_(meas) Emission-wavelength DR_(em)DR_(em, filtered)

The primary measurement of fluorescence is Flr_(meas), the intensity offluorescent light measured at the filtered detector.

The diffuse reflectance measurement Flr_(meas) represents thepropagation of photons to the non-filtered arm and is composed primarilyof excitation photons.

DR_(em) and DR_(em,filtered) represent the propagation of emission-onlyphotons.

Referring to Table 2, light intensity measured by the second (filtered)light detector 224 during illumination by the excitation-wavelengthlight captures the raw intensity of light emitted by the exogenousfluorescent agents (Flr_(meas)) prior to any corrections for tissueoptical properties in various aspects. After baseline subtractioncorrections as described herein previously, the emission-wavelengthlight contained in Flr_(meas) is assumed to originate predominantly fromthe exogenous fluorescent agent, with only minor contributions due toauto-fluorescence by endogenous chromophores, and is therefore termedFlr_(agent). In an aspect, if no change in the optical properties of thepatient's skin is assumed, all autofluorescence contributions would besubtracted off during the baseline correction described herein above.

However, if the optical properties of the patient's skin change duringthe acquisition of data, slightly more or less of the autofluorescencemay emerge from the patient's skin at the emission wavelength, therebyintroducing uncertainty into the accuracy of the background subtractioncorrection performed previously. In addition, varied skin opticalproperties may further alter the intensity of light at the excitationwavelength reaching the exogenous fluorescent agent, thereby alteringthe amount of energy absorbed by the exogenous fluorescent agent and theintensity of induced fluorescence from the exogenous fluorescent emittedin response to illumination by the excitation-wavelength light. Invarious aspects, the remaining three light measurements enablemonitoring of the optical properties of the patient's skin and providedata that may be used to adjust for any changes in the opticalproperties of the patient's skin.

Referring again to Table 2, the represented signals DRex_(meas) andFlr_(meas), which have been corrected for variations in temperature andoptical output are further processed to signals attributed only tophotons of the desired wavelength prior to applying a diffusereflectance correction. The number of photons due to either diffusereflectance, excitation or fluorescence on either detector depends onlight directionality and the gain of the detector at the detectedwavelength, as shown below:DRex _(meas) =A ₁ *DRex _(photons) +B ₁ *Flr _(photons)Flr _(meas) =A ₂ *DRex _(photons) +B ₂ *Flr _(photons)where the coefficients A1, A2, B1, B2 are composed of a directionalityand gain factor, e.g. A₁=d450_(SPM1)*G_(SPM1@450)

Isolation of the signals arising from fluorescence emission and diffusereflectance, excitation wavelength photons is performed as follows:

${{B_{1}\left( {\frac{B_{2}}{B_{1}} - \frac{A_{2}}{A_{1}}} \right)}{Flr}_{photons}} = {{Flr}_{meas} - {\frac{A_{2}}{A_{1}}{DR}_{{ex}_{meas}}}}$${{A_{2}\left( {\frac{A_{1}}{A_{2}} - \frac{B_{1}}{B_{2}}} \right)}{DRex}_{photons}} = {{DR}_{{ex}_{meas}} - {\frac{B_{1}}{B_{2}}{Flr}_{meas}}}$

Since the renal function monitor measures rate, which is independent ofmagnitude, the constant terms in front of the photons signals

$\left( {e.g.\mspace{14mu}{B_{1}\left( {\frac{B_{2}}{B_{1}} - \frac{A_{2}}{A_{1}}} \right)}} \right)$are not needed, as demonstrated below.

${IF} = {\left. {C_{0} + {C_{1}e^{{- t}/\tau}}}\rightarrow{\log({IF})} \right. = {{\log\left( C_{1} \right)} - \frac{1}{t}}}$

As such the terms

$\frac{A_{2}}{A_{1}}\left( {{or}\mspace{14mu} C_{ExLT}} \right)\mspace{14mu}{and}\mspace{14mu}\frac{B_{1}}{B_{2}}\left( {{or}\mspace{14mu} C_{FlrLT}} \right)$can be determined experimentally to isolate Flr_(photons) andDRex_(photons), respectively.

The table below represents the names of the signals used to representeach of the four measured signals in the diffuse reflectance correctiondevelopment. Note that either of the described preprocessing paths canbe followed to arrive at signals that can be used to develop thecorrection.

TABLE 3 Light Detector Measurements Used to Obtain FluorescenceMeasurements Corrected for Variable Tissue Optical Properties Genericsignal name Preprocessed signals for use DR_(ex) DR_(ex) _(photons) orDR_(ex) ₂ Flr Flr_(photons) or Flr₂ DR_(em) DR_(em) DR_(em, filtered)DR_(em, filtered)

-   -   where either of the excitation wavelength signals may be used as        alternate methods for obtaining a diffuse reflectance correction        with either of the described pre-processing methods.

Referring again to Table 2, light intensity measured by the first(unfiltered reference) light detector 222 during illumination byexcitation-wavelength light captures a measure of the diffusereflectance of excitation-wavelength light propagated through thepatient's skin (DR_(ex) _(meas) ). Although the first light detector 222is configured to detect both excitation-wavelength andemission-wavelength light, the intensity of the excitation-wavelengthlight is orders of magnitude higher than the intensity of theemission-wavelength light as a result of the lower efficiency ofproducing light via fluorescence. In various aspects, the proportion ofemission-wavelength light within DR_(ex) _(meas) is assumed to benegligible. In other aspects, the proportion of emission-wavelengthlight within DR_(ex) _(meas) is estimated and subtracted. Without beinglimited to any particular theory, because the intensity of theexcitation-wavelength light directed into the patient's skin is assumedto be relatively constant with negligible losses due to absorption bythe exogenous fluorescent agent, and is subject to power corrections asdescribed herein previously, DR_(ex) _(meas) serves as a benchmarkmeasurement to assess changes in the optical properties of the patient'sskin with respect to the excitation-wavelength light.

Light intensity measured by the first (unfiltered reference) lightdetector 222 during illumination by emission-wavelength light captures ameasure of the diffuse reflectance of emission-wavelength lightpropagated through the patient's skin (DR_(em)). Without being limitedto any particular theory, because the exogenous fluorescent agent is notinduced to emit emission-wavelength light due to the absence ofexcitation-wavelength illumination during this phase of the dataacquisition cycle, and because the intensity of the emission-wavelengthlight directed into the patient's skin is relatively constant andsubject to power corrections as described herein previously, DR_(em)serves as a benchmark measurement to assess changes in the opticalproperties of the patient's skin with respect to the emission-wavelengthlight.

Light intensity measured by the second (filtered) light detector 224during illumination by emission-wavelength light captures a secondmeasure of the diffuse reflectance of emission-wavelength lightpropagated through the patient's skin (DR_(em,filtered)). In one aspect,DR_(em,filtered) is subject to the same assumptions as DR_(em) asdescribed herein above. In addition, DR_(em,filtered) provides a meansof assessing heterogeneity of the tissue's optical properties. BecauseDR_(em,filtered) is measured by the second light detector 224 configuredto detect light emerging from the patient's skin at the third region 210(see FIG. 2), the intensity of light measured in DR_(em,filtered) haspropagated along an optical path through the skin of the patient that isdifferent from the optical path travelled by the light measured inDR_(em). Without being limited to any particular theory, because thedistances of the first detector aperture 1004 and second light aperture2006 through which light is delivered to the first and second lightdetectors 222/224, respectively are designed to be equidistant from thelight delivery aperture 1002 (see FIG. 10), any differences betweenDR_(em,filtered) and DR_(em) are assumed to be a result of heterogeneityon the optical properties of the skin traversed by the two differentoptical paths.

In one aspect, the intrinsic fluorescence (IF), defined here as themeasured fluorescence at the emission wavelength attributable only toemission by the exogenous fluorescent agent, may be calculated accordingto Eqn. (20):

$\begin{matrix}{{IF} = \frac{F_{meas}}{{DR}_{ex}^{k_{ex}}{DR}_{em}^{k_{em}}{DR}_{{em},{filtered}}^{k_{{em},{filtered}}}}} & {{Eqn}.\mspace{14mu}(20)}\end{matrix}$

The factors IF, Flr, DRex, DR_(em) and DR_(em,filtered) are definedherein above. As expressed in Eqn. (20), each of the diffuse reflectancecorrection measurement signals DR_(ex), DR_(em), and DR_(em,filtered)factors are raised to the powers k_(ex), k_(em), and k_(em,filtered)respectively. In an aspect, each measurement in Table 2 is subjected tothe power/temperature corrections and background subtraction correctionsas described herein above (see FIGS. 22A and 22B) before applying thediffuse reflectance correction of Eqn. (20).

In various aspects, the values of λ_(ex), k_(em), and k_(em,filtered)may be determined empirically. Non-limiting examples of suitableempirical methods for determining suitable values for λ_(ex), k_(em),and k_(em,filtered) include a global error map method and a linearregression method, both described in detail herein below.

In one aspect, once the values for each of the powers (k_(ex), k_(em),k_(em, filtered)) are identified, the same set of exponents may bereused for subsequent measurements of intrinsic fluorescence.Non-limiting examples of applications of the systems and methodsdescribed herein in which a set of selected exponents may be reusedincludes: repeated measurements on the same patient using the samesensor head 204; repeated measurements using the same sensor head ondifferent patients of the same species; repeated measurements usingdifferent sensor heads with the same design on patients of the samespecies; repeated measurements using different sensor heads withdifferent designs on patients of the same species; repeated measurementsusing different sensor heads with the different designs on patients ofthe different species; and any other suitable applications of thesystems and methods described herein. In another aspect, the exponentsmay be updated with repeated use of the systems and methods describedherein. In this other aspect, new sets of exponents may be determinedfor each use of the system and methods, and the stored sets of exponentsmay be periodically or continuously evaluated to assess whether anupdated selection of exponents is indicated. By way of non-limitingexample, if an analysis of multiple sets of exponents determined thatthe exponents did not vary outside of a threshold range in previous usesof the system, the system may be used to conduct measurements using theprevious set of exponents, a mean/median of all previous sets ofexponents, or any other estimate of suitable exponents based on previousvalues of exponents. In this non-limiting example, if an analysis ofmultiple sets of previous exponents determined that the exponents variedoutside of a threshold range, de novo selection of exponents using oneof the methods described herein below may be indicated.

Global Error Map Method

In one aspect, the values of the powers used in Eqn. (20) above aredetermined empirically using a global error surface method.

A flow chart illustrating the various steps of a global error surfacemethod 1400 is illustrated in FIG. 14. The method in this aspectincludes selecting ranges of values for each of the powers (k_(ex),k_(em), k_(em, filtered)) for each of the diffuse reflectance signals(DR_(ex), DR_(em), DR_(em,filtered)) are selected by a user at step1402. In various aspects, the ranges of values for each of the powersmay be influenced by any one or more of a variety of factors including,but not limited to: the design of the system 200, including the designof the sensor head 204; the properties of the selected exogenousfluorescent agent such as excitatory/emission wavelengths, absorptionefficiency, emission efficiency, and concentration of initial dose inthe patient's tissues; the species of the patient 202 and correspondingconcentrations of endogenous chromophores; the position of the sensorhead 204 on the patient 202; and any other relevant factor.

In one aspect, the method may include choosing a wide range for eachcoefficient (k_(ex), k_(em), k_(em,filtered)) and conduct a broadsearch. The error surfaces from this broad search may be analyzed tolocate wells in the error surface and the associated ranges for each ofthe coefficients. The method in this one aspect includes adapting theranges of each coefficient to include the regions from the broad searchwithin which wells in the error surface were observed and repeating theanalysis. This method may be iterated until a suitably fine resolutionis achieved that is capable of accurately capturing the minimum error.In one non-limiting example, for a human patient, the selected ranges ofpotential factors may be [0,2] for k_(ex), [0,4] for k_(em), and [−4,0]for k_(em,filtered).

Referring again to FIG. 14, step sizes may be selected at 1404 for theranges of values selected at 1402 for each power k_(ex), k_(em),k_(em, filtered). In an aspect, the step size for each factor may beselected based on any one or more of at least several factors including,but not limited to: the anticipated sensitivity of the IF valuescalculated by Eqn. (20) to changes in each factor; a suitable totalnumber of combinations of powers used to calculate IF considered factorsincluding available computational resources, acceptable data processingtimes, or any other relevant factors; and any other suitable criterionfor step size.

In various aspects, the step sizes may be the same value for all powersk_(ex), k_(em), k_(em, filtered). By way of non-limiting example, thestep size for all powers may be 0.5. In various other aspects, the stepsizes may be constant for all values of a single power k_(ex), k_(em),k_(em, filtered), but the step sizes selected for each power may bedifferent between different powers. By way of non-limiting example, theselected step size for k_(ex) may be 0.01 and the selected step size fork_(em) and k_(em,filtered) may be 0.6. In various additional aspects,the step size within one or more of the powers may vary within the rangeof values for each power. By way of non-limiting example, the selectedstep sizes for k_(ex) may be non-linearly distributed about the meanvalue. In this non-limiting example, the vector of potential values fork_(ex) may be [0 0.5 0.75 0.9 1 1.1 1.25 1.5 2]. In these variousadditional aspects, the step size may be reduced within subranges ofvalues for a power for which the IF calculated by Eqn. (20) is predictedto be more sensitive to small changes in that power. Non-limitingexamples of suitable varying step sizes within a range of values for asingle power include: different step sizes selected by a user, randomstep sizes, a linear increase and/or decrease in step size, a non-lineardistribution of different step sizes such as a logarithmic distribution,an exponential distribution, or any other suitable non-lineardistribution of step sizes.

Referring again to FIG. 14, the ranges of exponents selected at 1402,together with the step sizes selected at step 1404, may be used to formvectors of potential values of k_(ex), k_(en), k_(em, filtered) at 1406.By way of non-limiting example, assuming selected ranges of potentialexponents of [0,2] for k_(ex), [0,4] for k_(em), and [−4,0] fork_(em,filtered), and assuming a constant step size of 0.5 for allpowers, the vectors created at 1406 are:

k_(ex)=[0.0 0.5 1.0 1.5 2.0] (5 values)

k_(em)=[0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0] (9 values)

k_(em,filtered)=[−4.0 −3.5 −3.0 −2.5 −2.0 −1.5 −1.0 −0.5 −0.0] (9values)

Referring again to FIG. 14, for each combination of exponents amongstall vectors formed at 1406, IF is calculated from the measurements Flr,DR_(ex), DR_(em), and DR_(em,filtered) at 1408 using Eqn. (20). For eachcombination of exponents, a plurality of IF values are calculated at1408 in which each IF value corresponds to one of the data acquisitioncycles (i.e. a single sequence of emission-wavelength illuminationfollowed by excitatory-wavelength illumination as illustrated in FIG.5). By way of non-limiting example, using the vectors of potentialexponents listed herein above, a total of 405 (5*9*9) pluralities of IFsignals would be calculated.

In an aspect, the plurality of combinations of potential exponents maybe evaluated to select one combination of exponents from the pluralityto assign for use in subsequent diffuse reflectance correctionscalculated using Eqn. (20). Referring again to FIG. 14, an estimate oferror of the corrected Flr signal data (i.e. IF signal data calculatedusing Eqn. (20)) may be calculated at 1410. Any estimate of error may becalculated at 1410 including, but not limited to, a quantity related toresiduals of the IF signal data relative to a curve fit of the IF signaldata. Any type of known curve-fitting method may be used to curve-fitthe IF signal data including, but not limited to, a single-exponentialcurve fit. Without being limited to any particular theory, it is thoughtthat the rate of clearance of an exogenous fluorescent agent, such asMB-102, from the kidneys is expected to be a constant exponential decaycharacterized by the renal decay time constant RDTC.

In an aspect, a subset of the Fir signals corresponding to thepost-agent administration period 1508/1510 may be selected to estimatean error for each combination of exponents used to calculate the IFsignals using Eqn. (20) against a reference curve, including, but notlimited to, a curve obtained using plasma measurements. By way ofnon-limiting example, if the exogenous fluorescent agent is introducedinto the tissues of the patient by way of intravenous injection, thepost-agent administration period includes the period after injection inwhich the exogenous fluorescent agent has undergone sufficient diffusionfrom the blood into the extracellular fluid space throughout the patientso that the decay of the fluorescence is representative of clearance ofthe agent by the kidneys. In various aspects, the post-agentadministration period 1508/1510 of the Fir measurements may be selectedby any suitable method without limitation. Non-limiting examples ofsuitable methods for identifying a post-agent administration periodinclude: selection via inspection by a user and an automated selectionmethod such as the equilibration detection method enabled by theequilibrium selection subunit 1308 as described in detail herein below.

FIG. 15 is a graph of fluorescence measurements obtained from a patientover a period of about 10 hours after injection of an exogenousfluorescence agent (MB-102) after a pre-injection period of about 3hours. Referring to FIG. 15, the pre-injection/baseline period 1502 ischaracterized by a relatively low and stable fluorescence level, likelydue the absence of endogenous fluorescent agent in the blood of thepatient. After the injection 1503 of the exogenous fluorescence agent,the fluorescence measurements exhibit a sharp increase 1504 to a peakconcentration 1506, followed by a relatively smooth exponential decrease1508 back to background fluorescence levels as the kidneys eliminate theexogenous fluorescence agent from the blood of the patient. Withoutbeing limited to any particular theory, it is thought the injectedexogenous fluorescence agent is likely equilibrated across theextra-cellular space once the decay of the fluorescence iswell-described by a linear fit (or a line on semi-log plot). FIG. 16 isan enlargement of the graph of FIG. 15 showing a comparison of themeasured fluorescence data to a linear curve fit 1604 to the log of theIF signal within a portion of the post-equilibrium period 1510,demonstrating the close fit of the single-exponential curve-fit to theIF signal data.

In an aspect, the log of the calculated IF signal value may be fit witha line and an error of the curve fit relative to the individual IFvalues may be compared to the IF signals calculated using Eqn. (20) foreach of the plurality of combinations of exponents to calculate theerror at 1410. Any statistical summary parameter suitable forquantifying the error of the single-exponent curve fit and thecorresponding IF signal values may be used without limitation including,but not limited to: root mean square (RMS) error, average absolutedeviation, mean signed deviation, mean squared deviation, and any othersuitable statistical summary parameter. In one aspect, the calculatederror at 1410 may be the normalized RMS error of the linear fit of thelog(IF) signals. In this aspect, the normalized RMS error calculated at1410 is a single numerical quantity to facilitate the subsequentselection of a single combination of exponents from the plurality ofcombinations identified at 1406.

Referring again to FIG. 14, the method 1400 includes selecting a singlecombination of exponents at 1412 from among the plurality ofcombinations for which IF was calculated at 1408. Without being limitedto any particular theory, it is assumed that the combination ofexponents associated with a calculated IF signal that minimizes theerror calculated at 1410 is best suited for correcting the measured Flrsignals to remove the effects of variation in the optical properties ofthe patient's skin during data acquisition within the post-agentadministration period 1508/1510. In various aspects, any known method ofidentifying the combination of exponents may be used without limitationincluding, but not limited to, selecting the single combinations ofexponents from a map of all error values corresponding to allcombinations of exponents.

In various aspects, the plurality of error values corresponding to theplurality of combinations of exponents may be transformed into an errormap comprising a three-dimensional volume in which each of the threedimensions corresponds to the powers used in Eqn. (20): k_(ex), k_(em),and k_(em,filtered), respectively. In these various aspects, each errorvalue corresponding to one of the combinations of exponents is mapped toa coordinate (k_(ex1), k_(em1), and k_(em,filtered1)) within thethree-dimensional volume, where k_(ex1), k_(em1), and k_(em,filtered1)are the numerical values for one combination of exponents. In variousaspects, each error values may be mapped to the three-dimensional volumein any known format including, but not limited to: a number, a color, agreyscale value, and any other suitable format.

In an aspect, the three-dimensional map of error values described abovemay be transformed into a plurality of error surfaces corresponding to aplanar map of the error values associated with a single value of one ofthe powers k_(ex), k_(em), and k_(em,filtered), with the full numericalrange of the remaining two exponents acting as a horizontal axis and avertical axis of the error map.

FIG. 17 is an error map of the normalized RMS errors of thesingle-exponential curve-fits of the calculated IF signals maps to thefull ranges of k_(em,filtered) (horizontal axis) and k_(ex) (verticalaxis) at a constant value of k_(em) in which the normalized RMS errorsare represented as colors on the error map. In one aspect, thenormalized RMS value calculated for each coefficient may be normalizedaccording to Eqn. (21):

$\begin{matrix}{{RMSE}_{CV} = \sqrt{\sum\left\lbrack \frac{{IF}_{agent} - {{fit}\left( {IF}_{agent} \right)}^{2}}{{fit}\left( {IF}_{agent} \right)} \right\rbrack}} & {{Eqn}.\mspace{14mu}(21)}\end{matrix}$in which IF_(agent) is the calculated IF signal, and fit(IF_(agent)) isthe corresponding value of the single-coefficient curve fit equation. Inone aspect, the global error map method of determining the powers to beused in the correction for variation in skin optical properties byanalyzing a single measurement set as described herein above. In anotheraspect, the global error map method may analyze and combine multiplemeasurement datasets for multiple individuals obtained using the samesystem and/or sensor head. In yet another aspect, the global error mapmethod may analyze multiple measurement datasets from multipleindividuals obtained using multiple systems and sensor heads. In anaspect, the powers to be used in the correction according to Eqn. (20)may be determined for each measurement of each individual. In otheraspects, the powers to be used may be obtained using at least severaldifferent measurement datasets and the powers so obtained may be storedfor subsequent use for measurement datasets obtained from a newindividual and or obtained using a different system and/or sensor. Invarious aspects, the projection across each of the error surfaces (ak_(em,filtered)−k_(ex) projection, a k_(em,filtered)−k_(em) projection,and a k_(em)−k_(ex) projection) may be inspected to determine whetherthe power ranges defined at 1402 were adequate. In one aspect, the errorsurface may be inspected to confirm that the map includes a clearlydefined minimum value. In this one aspect, if the inspection of theerror map does not identify a minimum value, the ranges of one or morepower vales may be revised and the method 1400 may be repeated. In oneaspect, an assessment of the optical properties of the skin of thepatient (e.g. melanin absorbance, blood content, and/or scatteringcoefficient) may be used to categorize the patient so that anappropriate set of coefficients may be selected for that category.

In an aspect, the combination of exponents k_(ex), k_(em), andk_(em,filtered) may be stored and used for subsequent measurementsconducted by the system 200. FIG. 18 is a graph comparing the rawfluorescence signal (blue line) to the calculated IF signal (red line)for a measurement data set. In an additional aspect, a global correctionmay be calculated by combining measurements obtained using a pluralityof different systems and/or sensor heads and identifying the combinationof exponents corresponding to an overall minimum error value.

Linear Regression Method

In one aspect, the values of the power coefficients used in Eqn. (20)above are determined empirically using a linear regression method. Aflow chart illustrating the various steps of the linear regressionmethod of obtaining a correction in the form of a regression equationwith predictor variables (DR_(ex), DR_(em), DR_(em,filtered)) isprovided in FIG. 19,

Referring to FIG. 19, the method 1900 may include log transforming Flrto log (Flr) to prepare the raw fluorescence measurements Fr foranalysis at 1902. FIG. 20 is a graph of log (Flr) produced at 1902.Referring again to FIG. 19, the method 1900 may further includeselecting a region of stable optical properties 2002 (see FIG. 20) in anaspect, In this aspect, regions of stable optical properties 2002typically correspond to linear segments on the graph of log (Flr) asshown in FIG. 20. In this aspect, the method 1900 further includesobtaining a linear regression model 2004 within the region of stableoptical properties 2002 at 1906. The linear regression model 2004 may beobtained using any regression method without limitation including, butnot limited to, a multi-variable linear regression modeling method.

Referring again to FIG. 19, the method 1900 may further includeextending the linear regression model 2004 obtained within the region ofstable optical properties 2002 to produce an extended linear regression2008 extending into a region of variable optical properties 2010 at1908. In an aspect, the region of variable optical properties 2010 ischaracterized by a non-linear profile within the graph of log (Flr) asillustrated in FIG. 20.

Referring again to FIG. 19, the method 1900 may further includeobtaining a linear regression model 2004 with predictor variables Flr,DR_(ex), DR_(em), and DR_(em,filtered) at 1910 and the linear curve fit2004 as the predicted response. The extension of the linear regression2008 produced at 1908 may be used to train the linear regression modelobtained at 1910.

The linear regression model may be developed using a measurement dataset obtained from a single individual and/or a single system and sensorhead in one aspect. In another aspect, the linear regression model maybe developed using multiple measurement datasets obtained from multipleindividuals and/or multiple systems and sensor heads. In some aspects, alinear regression model may be developed de novo for each newmeasurement data set obtained for an individual. In at least some otheraspects, the constants and parameters characterizing a linear regressionmodel developed as described herein above may be stored for subsequentuse in lieu of developing a linear regression model de novo for eachmeasurement data set obtained as described herein above.

d) Fault Detection Subunit

Referring again to FIG. 13, the processing unit 236 of the controller212 may further include a fault detection subunit 1312 configured tomonitor the function of the light sources 218/220 and light detectors222/224 and to inform the user of any irregularities of any detectedfaults within the system 200 via the display unit 216. In variousaspects, the fault detection subunit 1312 may enable the basicidentification of fault and notice states by examining the signal levelsreceived from the light sources 218/220 and light detectors 222/224 andassociated additional temperature sensors 228 and additional lightdetectors 226 of the sensor head 204 (see FIG. 2). In various aspects,the signal magnitudes (see Eqn. (1)) and average signals may be used todetermine the peak and nadir levels of the modulation of the LED lightsources 218/220. The nadir of the signal, defined herein as the averagesignal minus half the peak-to-peak signal, may be used to monitorambient light levels in one aspect. Without being limited to anyparticular theory, additional contributions to the nadir levels of themodulated signals, such as amplifier DC offset, may be neglected assmall and constant relative to the contributions of ambient lightleakage. In an aspect, if the detected ambient light levels register inexcess of about one quarter of the high-speed ADC 1102 range at lowdetector amplifier gain, an ambient light notice is issued to the uservia the display unit 216.

In various other aspects, saturation of the light detectors 222/224detectors may also be monitored by the fault detection subunit 1312. Inthese other aspects, the saturation may be monitored by calculating thepeak value of the signal, defined herein as the average signal valueplus half the peak-to-peak signal. If the signal's peak value fallswithin is within 5% of saturation of the ADC range, the fault detectionsubunit 1312 may issue a saturation notice to the user via the displayunit 216. If saturation event is detected by the fault detection subunit1312, the ambient light level may then be checked to determine if thesaturation event is associated with ambient light saturation, definedherein as a saturation event occurring concurrently with an ambientlight notice as described herein above. If an ambient light saturationevent is detected, the fault detection subunit 1312 issues an ambientlight saturation notice to the user via the display unit 216, and dataacquisition by the acquisition unit 234 is continued in this noticestate to allow the user to resolve the condition. If a saturation eventis detected that is not associated with an excess of ambient light, thefault detection unit may signal the light detector control unit 232 toperform an adjustment of detector gain and/or may signal the lightsource control unit 230 to perform an adjustment to the LED currentsource 1126 to adjust LED intensity. In various aspects, the faultdetection unit issues a notification to the user via the display unit toreport either the ambient light saturation event, or the saturationevent not associated with an excess of ambient light. In some aspects,if a saturation event is detected, but the automatic gain adjustment hasbeen disabled by a user when the system 200 is configured in theEngineering Mode as described herein above, the user is also notifiedvia the display unit.

e) Post-Agent Administration Selection Subunit

Referring again to FIG. 13, the processing unit 236 may further includea post-agent administration selection subunit 1308 configured toautomatically identify the portion of the measurement data set thatcorresponds to the post-agent administration period 1508/1510 (see FIG.15). Referring against to FIG. 15, as described herein above, after anexogenous fluorescent agent, such as MB-102, is injected into thebloodstream of a patient, the exogenous fluorescent agent undergoes anequilibration period of diffusion from the bloodstream into the rest ofthe extracellular tissues of the patient. After the agent injection1503, the temporal profile of the fluorescence signal Flr may becharacterized as a two-exponential signal profile described by Eqn.(22):IF _(pre-equilibration) =C ₀ +C ₁ e ^(−τ/τ) ¹ +C ₂ e ^(−t/τ) ²   Eqn.(22)in which C₀ is the baseline signal that is typically removed by baselinesubtraction as described herein above.

Referring again to FIG. 15, once the diffusion of the exogenousfluorescent agent into the extracellular tissues of the patient reachesa quasi-steady state condition, post-equilibration period 1510 isachieved and the fluorescence signal may be characterized as a lineardecay. Without being limited to any particular theory, thepost-equilibration region of the measurement data set is assumed to becharacterized as a region of the IF temporal profile that, whenlog-transformed, is well-described by a linear equation. In one aspect,the post-equilibration region is well-described described by Eqn. (23):IF _(post-equilibration) =C ₀ +C ₁ e ^(−t/τ)  Eqn. (23)

In an aspect, the post-agent administration selection subunit 1308 mayidentify the post-agent administration period 1510 automatically byperforming a single-exponent curve fit at different portions of the IFdata set and analyzing the associated curve fitting errors for each ofthe different portions. In various aspects, the post-agentadministration selection subunit 1308 may select the earliest-occurringportion of the IF data set in which the curve-fit error associated witha single-exponent curve fit falls below a threshold value as the initialpost-agent administration portion of the IF data set suitable for datacorrection and analysis as described herein above. Any analysis methodsuitable for comparing curve-fit errors association withsingle-exponential curve fits of different portions of the IF data setmay be used in the post-agent administration selection subunit 1308including, but not limited to, linear curve-fitting portions of the IFdata set falling within overlapping or non-overlapping data windows andcomparing the curve-fit errors of the corresponding data windows. In anaspect, the post-agent administration selection subunit 1308 may produceat least one signal configured to signal the time range within the IFdata set corresponding to the post-agent administration period 1508/1510to the diffuse reflectance correction subunit 1306 and/or RDTCcalculation subunit 1310 to enable the selection of a suitable portionof the IF data set to correct and analyze as disclosed herein.

In another aspect, a linear fit and a 2-exponential fits to the IF datamay be compared. In this other aspect, equilibration may be identifiedas complete once the fitting error is equivalent (corrected for theextra degrees of freedom in the 2-exponential fit).

f) RDTC Calculation Subunit

In various aspects, the system 200 is configured to transform thevarious measurements from the light detectors 222/224 and associatedlight sources 218/220 and other thermal and light sensors into acorrected intrinsic fluorescent (IF) signal corresponding to thedetected fluorescence attributable solely to emission of fluorescence bythe exogenous fluorescent agent at the emission wavelength in responseto illumination by light at the excitatory wavelength. In variousaspects, the exponential decrease of the IF signals during thepost-agent administration portion of the IF data set may be analyzed tomonitor and quantify renal function.

In one aspect, the exponential decrease of the IF signals during thepost-agent administration portion of the IF data set may be transformedinto a glomerular filtration rate (GFR) configured to quantify renalfunction. In another aspect, the exponential decrease of the IF signalsduring the post-equilibration portion of the IF data set may betransformed into a renal decay time constant (RDTC), also configured toquantify renal function. In another aspect, the exponential decrease ofthe IF signals during the post-equilibration portion of the IF data setmay be transformed into a renal decay rate, also configured to quantifyrenal function.

Referring again to FIG. 13, the processing unit 236 may further includean RDTC calculation subunit 1310 configured to automatically transformthe IF signals into a renal decay time constant (RDTC). As used herein,renal decay time constant (RDTC) is defined as the time constantassociated with the post-equilibration single-exponential decaydescribed in Eqn. (23) herein above. In one aspect, after accuratebaseline subtraction by the baseline subtraction subunit 1304, the renaldecay time constant τ may be calculated by performing a linearregression on the log-transformed IF signal data (log (IF)), asdescribed in Eqn. (24):

$\begin{matrix}{{\log({IF})} = {{\log\left( C_{1} \right)} - {\frac{1}{\tau}t}}} & {{Eqn}.\mspace{14mu}(24)}\end{matrix}$

In various aspects the RDTC calculation subunit 1310 may produce signalsconfigured to produce a display of the calculated RDTC using the displayunit 216. The display of the calculated RDTC may be provided to thedisplay unit 216 in any suitable format including, but not limited to: agraph of RDTC as a function of time, a single discrete RDTC value, atable of RDTC values as a function of time, a color-coded display orother graphical representation configured to specific whether thecalculated RDTC may be classified as normal/healthy, abnormal, high,low, and any other suitable classification. In various other aspects,any of the graphical formats described above may be continuously ornon-continuously updates as additional data is obtained and analyzed. Inone aspect, the RDTC calculation subunit 1310 may calculate RDTC asdescribed herein above within non-overlapping and/or overlapping windowswithin the IF data set.

In another aspect, the RDTC calculation subunit 1310 may convert RDTCinto glomerular filtration rate (GFR) using known methods. In thisaspect, RDTC may be inverted and multiplied by a slope, resulting incGFR, a prediction of GFR that may be corrected for body size (e.g. bodysurface area, or volume of distribution).

v) Memory

Referring again to FIG. 2, the controller 212 of the system 200 mayfurther include a memory 242 configured to facilitate data storage inthe system 200. In some embodiments, the memory 242 includes a pluralityof storage components such as, but not limited to, a hard disk drive,flash memory, random access memory, and a magnetic or optical disk.Alternatively or additionally, the memory 242 may include remote storagesuch a server in communication with the controller 212. The memory 242stores at least one computer program that, when received by the at leastone processor, cause the at least one processor to perform any of thefunctions of the controller 212 described above. In one implementation,the memory 242 may be or contain a computer-readable medium, such as afloppy disk device, a hard disk device, an optical disk device, or atape device, a flash memory or other similar solid state memory device,or an array of devices, including devices in a storage area network orother configurations. A computer program product can be tangiblyembodied in an information carrier. The computer program product mayalso contain instructions that, when executed, perform one or morefunctions, such as those described herein. The information carrier maybe a non-transitory computer- or machine-readable medium, such as thememory 242 or memory on the processor 238.

In various aspects, the system 200 may record raw measurements andprocessed data to a series of files. Each file may contain a header,which contains information about the operator, instrument, and session.Each experimental session records a set of files into a separate folderfor each sensor head used in that session. The raw data file maycontains in-phase, quadrature, and average measurements from thedetectors and monitors during the active periods of both the excitationwavelength and the emission wavelength LEDs, along with the gainsettings of the LEDs and detectors at the time of data acquisition.

In various other aspects, the processed data file may contain thefluorescence and diffuse reflectance measurements after magnitudecalculation and correction for the monitor readings, along with the gainsettings of the LEDs and detectors. The intrinsic fluorescence data filemay contain the intrinsic fluorescence measurements resulting from thediffuse reflectance correction of the raw fluorescence signals. The GFRfile may contain the calculated GFR as a function of time, classified toindicate whether post-equilibration has occurred, along with confidencebounds. The telemetry file may contain the temperature and voltagemeasurements. The event record file may contain both user andautomatically generated event records.

vi) GUI Unit

Referring again to FIG. 2, the controller 212 may include a GUI unit 240configured to receive a plurality of signals encoding various measuredand transformed data from other units of the system in various aspects.In addition, the GUI unit may be configured to produce signalsconfigured to operate the display unit 216 in order to display data,frames, forms, and/or any other communications of information betweenthe user and the system 200.

vii) Processor

Referring again to FIG. 2, the controller 212 may further include aprocessor 238. The processor 238 may include any type of conventionalprocessor, microprocessor, or processing logic that interprets andexecutes instructions. The processor 238 may be configured to processinstructions for execution within the controller 212, includinginstructions stored in the memory 242 to display graphical informationfor a GUI on an external input/output device, such as display unit 216coupled to a high speed interface. In other implementations, multipleprocessors and/or multiple buses may be used, as appropriate, along withmultiple memories and types of memory. Also, multiple controllers 212may be connected, with each device providing portions of the necessaryoperations to enable the functions of the system 200. In someembodiments, the processor 238 may include the acquisition unit 234, thelight detector control unit 232, the light source control unit 230,and/or the processing unit 236.

As used herein, a processor such as the processor 238 may include anyprogrammable system including systems using micro-controllers, reducedinstruction set circuits (RISC), application specific integratedcircuits (ASICs), logic circuits, and any other circuit or processorcapable of executing the functions described herein. The above examplesare example only, and are thus not intended to limit in any way thedefinition and/or meaning of the term “processor.”

As described herein, computing devices and computer systems include aprocessor and a memory. However, any processor in a computer devicereferred to herein may also refer to one or more processors wherein theprocessor may be in one computing device or a plurality of computingdevices acting in parallel. Additionally, any memory in a computerdevice referred to herein may also refer to one or more memories whereinthe memories may be in one computing device or a plurality of computingdevices acting in parallel.

C. Operation Unit

The operation unit 214 may be configured to enable a user to interface(e.g., visual, audio, touch, button presses, stylus taps, etc.) with thecontroller 212 to control the operation of the system 200. In someembodiments, the operation unit 214 may be further coupled to eachsensor head 204 to control the operation of each sensor head 204.

D. Display Unit

Referring again to FIG. 2, the system 200 may further include a displayunit 216 configured to enable a user to view data and controlinformation of the system 200. The display unit 216 may further becoupled to other components of the system 200 such as the sensor head204. The display unit 216 may include a visual display such as a cathoderay tube (CRT) display, liquid crystal display (LCD), light emittingdiode (LED) display, or “electronic ink” display. In some embodiments,the display unit 216 may be configured to present a graphical userinterface (e.g., a web browser and/or a client application) to the user.A graphical user interface may include, for example, an display for GFRvalues as described herein above as produced by the system 200, andoperational data of the system 200

Exogenous Markers

Without being limited to any particular theory, molecules which arehighly hydrophilic and small (creatinine, molecular weight=113) tomoderately sized (inulin, molecular weight ˜5500) are known to berapidly cleared from systemic circulation by glomerular filtration. Inaddition to these properties, an ideal GFR agent would not be reabsorbednor secreted by the renal tubule, would exhibit negligible binding toplasma proteins, and would have very low toxicity. In order to designoptical probes that satisfy all of these requirements a balance wasstruck between photophysical properties, and the molecular size andhydrophilicity of the fluorophore. For example, while hydrophobiccyanine and indocyanine dyes absorb and emit optimally within the nearinfrared (NIR) biological window (700-900 nm), hydrophilicity is notsufficiently high to function as pure GFR agents. Smaller dye moleculesmay be more easily converted to the extremely hydrophilic speciesrequired for renal clearance, but the limited 7I-systems resulting fromthese lower molecular weight compounds generally enable one photonexcitation and emission in the ultraviolet (UV).

To resolve the pharmacokinetic issues in concert with enhancing thephotophysical properties, simple derivatives of2,5-diaminopyrazine-3,6-dicarboxylic acid act as very low molecularweight fluorescent scaffold systems with bright emission in theyellow-to-red region of the electromagnetic spectrum. SAR studies havebeen carried out using amide-linked variants of these derivatives forthe simultaneous optimization of GFR pharmacokinetics and photophysicalproperties. A variety of hydrophilic functionalities for enabling rapidrenal clearance of this class of pyrazine fluorophores includingcarbohydrate, alcohol, amino acid and various PEG-based linkerstrategies may be employed. PEG substitution maybe used to increasehydrophilicity and solubility, reduce toxicity, and modulate aggregationof the resulting pyrazine derivatives. Variations of molecular weightand architecture (and hence hydrodynamic volume) in a series ofmoderately sized PEG-pyrazine derivatives may also be suitable for useas endogenous fluorescent agents.

In one aspect, the exogenous fluorescent agent is MB-102.

EXAMPLES

The following example illustrates various aspects of the disclosedsystems and methods.

Example 1: Perturbation Analysis

To demonstrate the effectiveness of the diffuse reflectance datacorrection method described herein above, the following experiments wereconducted.

A system similar to the system 200 described herein above was used tomonitor the fluorescence produced during the renal elimination of anexogenous fluorescent agent, MB-102, using the methods described hereinabove, in particular the diffuse reflectance data correction method.

FIG. 21A is a graph summarizing the changes in the magnitude of the rawfluorescence signal (Flr) just prior to injection of the MB-102fluorescent agent into a pig and for about six hours post-injection.During the post-equilibration portion, corresponding to a time of about13:45 in FIG. 21A, the pig was subjected to a series of perturbationsselected to vary the optical properties of the pig's skin and/orunderlying tissues: administration of blood pressure medications toinduce vasodilation/vasorestriction 2102, application of pressure tocompress the tissue 2104, lateral movement of the sensor head 2106, SpO₂decrease 2108, SpO₂ decrease 2110, remove/replace sensor head 2112/2114,and skin cooling 2116.

FIG. 21B is a graph summarizing the corrected intrinsic fluorescencesignal (IF) corrected as described herein above with no baselinesubtraction. At time points later than 2 hours after agent injection,the time course of the IF signal was characterized by the expectedsingle-exponential decay of the signal, with attenuated variation due tothe applied perturbations.

Table 4 summarizes the specific effects of the diffuse reflectance datacorrection method on the Fir data associated with each individualperturbation:

TABLE 4 Effect of Diffuse Reflectance Data Correction PERTURBATIONEFFECT OF CORRECTION Pressure Application Decreased dataexcursions/outliers Lateral Sensor Movement Decreased dataexcursions/outliers SpO₂ Decrease Slope of IF signal decrease improvedSpO₂ Increase Slope of IF signal decrease improved Remove/Replace SensorHead Decreased data excursions/outliers Cooling No noticeable impact

FIG. 21C is a graph summarizing the detected diffuse reflectance signalsDR_(em,filtered), DR_(em), and DR_(ex) substituted into Eqn. (20) todetermine the diffuse reflectance correction of the raw Fir signal asdescribed herein above. As illustrated in FIG. 21C, the DR_(em,filtered)signal was the most sensitive to the various perturbations. The DR_(em),and DR_(ex) signals exhibited modest variation in response to theperturbations.

The results of these experiments demonstrated that the diffusereflectance data correction was capable of correcting the rawfluorescent signal data to compensate for the effects of a variety ofperturbations that induced a variety of changes in the skin's opticalproperties.

Example 2: Sensor Head with Flared Housing

FIG. 23 is a perspective view of a sensor head 204 a in another aspect.

In this other aspect, the sensor head 204 a includes a housing 600 aformed from an upper housing 602 a and a flared lower housing 604 a. Thesurface area of the lower housing 604 a expands to form an enlargedbottom surface 608 a. The housing 600 a further includes a cable opening806 a formed through the upper housing 602 a.

FIG. 24 is a bottom view of the sensor head 204 a showing the bottomsurface 608 a of the housing 600 a. The bottom surface 608 a may includean aperture plate 702 a including one or more apertures 704 a configuredto transmit light between the skin of the patient and the light sourcesand light detectors contained inside the housing 600. As illustrated inFIG. 24, the apertures 704 a include a light delivery aperture 1002 aconfigured to deliver illumination produced by the first and secondlight sources 218/220 to tissues of the patient 202, as well as firstand second detector apertures 1004/1006 configured to receive light fromthe tissues of the patient 202. In one aspect, the bottom surface 608 aenables the positioning of the apertures 704 a beneath a relativelylarge area obscured from ambient light conditions by the bottom surface608 a. This reduction of scattered ambient light entering the first andsecond detector apertures 1004/1006 reduces noise introduced into thelight intensity measurements obtained by the first and second lightdetectors 222/224.

In various aspects, the bottom surface 608 a of the housing 600 a may beattached the patient's skin using a biocompatible and transparentadhesive material 610 a including, but not limited to, a cleardouble-sided medical grade adhesive, as illustrated in FIG. 24. Thetransparent adhesive material 610 a may be positioned on the bottomsurface 608 a such that the adhesive material 610 a covers the apertures704 a.

FIG. 25 is an isometric view of the sensor head 204 a with the upperhousing 602 a and various electrical components removed to expose aninner housing 2502. FIG. 26 is an exploded view of the inner housing2502 and associated electrical components illustrated in FIG. 25.Referring to FIG. 25 and FIG. 26, the inner housing 2502 is containedwithin the housing 600 a and is mounted to the lower housing 608 a. Theinner housing 2502 contains a sensor mount 912 with a first detectionwell 908, a second detection well 910, and a light source well 902formed therethrough. The first light detector 222 is mounted within thefirst detection well 908 and the second light detector 224 is mountedwithin the second detection well 910. The first and second light sources218/220 are mounted within the light source well 902. In an aspect, thefirst detection well 908, second detection well 910, and light sourcewell 902 of the sensor mount 912 are optically isolated from one anotherto ensure that light from the light sources 218/220 does not reach thelight detectors 222/224 without coupling through the skin of the patient202. The separation between the two detection wells 908/910 ensures thatthe detected fluorescence signal from the exogenous fluorescent agent isdistinguishable from the unfiltered excitation light, as described indetail above.

Referring to FIG. 26, the inner housing 2502 includes a first detectionaperture 2602, second detection aperture 2604, and light source aperture2606. The sensor mount 912 is coupled to the inner housing 2502 so thatthe first detection aperture 2602, second detection aperture 2604, andlight source aperture 2606 are aligned with the first detection well908, second detection well 910, and light source well 902 of the sensormount 912, respectively.

In one aspect, optically transparent windows 2610, 2612, and 2614 arecoupled within first detection aperture 2602, second detection aperture2604, and light source aperture 2606, respectively, to seal theapertures while also providing optically transparent conduits betweenthe tissues and the interior of the sensor head 204 a. In addition,diffusers 2616, 2618, and 2620 are coupled over optically transparentwindows 2610, 2612, and 2614, respectively. The diffusers 2616, 2618,and 2620 are provided to spatially homogenize light delivered to thetissues by light sources 218/220 and to spatially homogenize lightdetected by light detectors 222/224. In an aspect, the absorption filter244 is coupled to the diffuser 2616. In one aspect, an opticallytransparent adhesive is used to couple the absorption filter 244 iscoupled to the diffuser 2616.

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and systems withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

When introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

What is claimed is:
 1. A method of monitoring a time-varyingfluorescence signal emitted from a fluorescent agent from within adiffuse reflecting medium with time-varying optical properties, themethod comprising: providing a measurement data set comprising aplurality of measurement entries, each measurement data entry comprisingat least two measurements obtained at one data acquisition time from apatient before and after administration of the fluorescent agent, the atleast two measurements selected from: a DR_(ex) signal detected at asecond region adjacent to the diffuse reflecting medium by an unfilteredlight detector during illumination of the diffuse reflecting medium byexcitatory-wavelength light from a first region adjacent to the diffusereflecting medium; an Flr signal detected at a third region adjacent tothe diffuse reflecting medium by a filtered light detector duringillumination of the diffuse reflecting medium by excitatory-wavelengthlight from the first region; and a DR_(em) signal detected at the secondregion by the unfiltered light detector during illumination of thediffuse reflecting medium by emission-wavelength light from the firstregion; identifying a post-agent administration portion of themeasurement data set; and transforming each Flr signal of eachmeasurement data entry within the post-agent administration portion ofthe measurement data set to an IF signal representing a detectedfluorescence intensity emitted solely by the fluorescent agent fromwithin the diffuse reflecting medium, wherein transforming comprisescombining the at least two measurements according to a transformationrelation comprising a mathematical equation converting Flr to IF.
 2. Themethod of claim 1, wherein the transformation relation consists of Eqn.(20): $\begin{matrix}{{{IF} = \frac{Flr}{{DR}_{{ex},{meas}}^{k_{{ex},{meas}}}{DR}_{em}^{k_{em}}{DR}_{{em},{filtered}}^{k_{{em},{filtered}}}}};} & {{Eqn}.\mspace{14mu}(20)}\end{matrix}$ wherein IF represents a fluorescence intensity emittedsolely by the fluorescent agent, and k_(ex,meas), k_(em), andk_(em,filtered) are exponents of terms DR_(ex), DR_(em), andDR_(em,filtered), respectively.
 3. The method of claim 2, whereink_(ex,meas), k_(em), and k_(em,filtered) are previously determined froma prior analysis of a prior measurement data set.
 4. The method of claim2, wherein k_(ex,meas), k_(em), and k_(em,filtered) are determined by aglobal error map method comprising: forming three vectors of proposedexponent values, each of three vectors comprising a plurality ofproposed values for k_(ex,meas), k_(em), and k_(em,filtered),respectively, transforming each Fir signal of each measurement dataentry within the post-agent administration portion of the measurementdata according to the transformation relation using each combination ofproposed values from the three vectors to form a plurality oftransformed data measurement sets; performing a single-exponential curvefit over at least a portion of the measurement data entries for each ofthe plurality of the transformed data measurement sets to obtain aplurality of curve-fit errors, each curve-fit error corresponding to onecombination of the proposed exponent values from the three vectors;assembling an error map comprising at least a portion of the pluralityof curve-fit errors mapped to a volume defined by two or more orthogonalaxes, each orthogonal axis comprising a range of proposed exponentvalues from one of the three vectors; identifying a minimum curve-fiterror within the error map; and selecting the proposed exponent valuescorresponding to the minimum curve-fit error for use in Eqn. (20). 5.The method of claim 4, wherein each of the plurality of curve-fit errorsconsists of a normalized root-mean-square fitting error of thesingle-exponential curve-fit.
 6. The method of claim 1, wherein thetransformation relation consists of a linear regression model formedwith predictor variables DR_(ex), DR_(em), and DR_(em,filtered) using alow-variability portion of the plurality of measurement data entriescharacterized by a curve-fit error falling below a threshold value for asingle-exponential curve-fit of the measurement data entries of the lowvariability portion.
 7. The method of claim 6, wherein the linearregression model is extrapolated to measurement data entries outside ofthe low variability region.
 8. The method of claim 1, further comprisingsubtracting a baseline value for Flr from each of the Flrvalues from theplurality of the measurement data sets prior to transforming eachFlrsignal of each measurement data entry within the post-agentadministration portion of the measurement data set to an IF signal. 9.The method of claim 1, wherein the at least two measurements are furtherselected from a DR_(em,filtered) signal detected at the third region bythe filtered light detector during illumination of the diffusereflecting medium by emission-wavelength light from the first region.10. The method of claim 2, wherein any one or more of k_(ex,meas),k_(em), and k_(em,filtered) is equal to 0.